Immunocompatible amniotic membrane products

ABSTRACT

Provided herein is a placental product comprising an immunocompatible amniotic membrane. Such placental products can be cryopreserved and contain viable therapeutic cells after thawing. The placental product of the present invention is useful in treating a patient with a tissue injury (e.g. wound or burn) by applying the placental product to the injury. Similar application is useful with ligament and tendon repair and for engraftment procedures such as bone engraftment.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/030,551 filed on Feb. 18, 2011, and which claims priority to:

U.S. Provisional Application Ser. No. 61/338,464 entitled “SelectivelyImmunodepleted Chorionic Membranes”, filed on Feb. 18, 2010,

U.S. Provisional Application Ser. No. 61/338,489 entitled “SelectivelyImmunodepleted Amniotic Membranes”, filed on Feb. 18, 2010, and

U.S. Provisional Application Ser. No. 61/369,562 entitled “TherapeuticProducts Comprising Vitalized Placental Dispersions filed on Jul. 30,2010, the contents of which are hereby incorporated by reference intheir entireties.

This application incorporates by reference, applications filed on Feb.18, 2011 entitled:

“Methods of Manufacture of Immunocompatible Chorionic MembraneProducts”, which received U.S. application Ser. No. 13/030,539,

“Immunocompatible Chorionic Membrane Products”, which received U.S.application Ser. No. 13/030,507,

“Methods of Manufacture of Immunocompatible Amniotic Membrane Products”,which received U.S. application Ser. No. 13/030,562,

“Therapeutic Products Comprising Vitalized Placental Dispersions”, whichreceived U.S. application Ser. No. 13/030,580, and

“Methods of Manufacture of Therapeutic Products Comprising VitalizedPlacental Dispersions”, which received U.S. application Ser. No.13/030,595.

FIELD OF THE INVENTION

The present technology relates to products to facilitate wound healingsuch as placenta membrane-derived products and biologic skin. Thepresent technology relates to products to protect injured or damagedtissue, or as a covering to prevent adhesions, to exclude bacteria, toinhibit bacterial activity, or to promote healing or growth of tissue.An example of such a placental membrane is an amniotic membrane. Thefield also relates to methods of manufacturing and methods of use ofsuch membrane-derived products.

BACKGROUND OF THE INVENTION

Fresh or decellularized placental membranes have been used topically insurgical applications since at least 1910 when Johns Hopkins Hospitalreported the use of placental membrane for dermal applications.Subsequently unseparated amnion and chorion were used as skinsubstitutes to treat burned or ulcerated surfaces. During the 1950's and60's Troensegaard-Hansen applied boiled amniotic membranes to chronicleg ulcers.

The human amniotic membrane (AM) is the innermost of the fetal membranesderiving from the amniotic sac and constituting the lining of theamniotic cavity. It is approximately 0.02 to 0.5 mm thick. The AMconsists of five layers: a thin layer rests on the basement membrane andcontacts the amniotic fluid, an underlying layer of connective tissueattaching the basement membrane that consists of three layers: a compactlayer, a layer of fibroblast, and a spongy layer. The spongy layer isadjacent to the cellular layer of the chorion. The amnion is essentiallydevoid of vasculature.

Both fresh and frozen AMs have been used for wound healing therapy. Whenfresh AM is used, there is increased risk of disease transmission.According to published reports, fresh amniotic tissue exhibits cellviability of 100%, however within 28 days of storage above 0° C.diminished cell viability to 15 to 35%. Freezing over a time of 3 weeksreduced cell viability to 13 to 18%, regardless of the temperature ormedium.

Lee and Tseng report the successful cryopreservation of AM in glyceroland Dulbeccos Modified Eagle medium (DMEM) at −80° C., although suchcryopreservation dramatically decreases cell viability. Thecryopreservation of AM in glycerol and DMEM is recommended by the FDA.According to published reports, glycerol storage of AM resulted inimmediate cell death. Glycerol cryopreserved AM (−80° C.) andglycerol-preserved AM (−4° C.) are sufficient to provide a matrix forwound healing, but fail to provide sufficient cell viability to bestowbiological effectiveness

Gajiwala and Gajiwala report the successful preservation of AM byfreeze-drying (lyophilisation) and gamma-irradiation. According to thismethod, AM is pasteurized at 60° C., treated with 70% ethanol, andfreeze-dried to remove most of the remaining moisture. Then the AM issterilized by exposure of 25 kGy gamma-radiation in a Cobalt 60 Gammachamber unit or at an ISO-certified radiation plant. The sterilized AMcan be stored at room temperature for a short period (up to 6 months).

Gomes reports preservation of AM with lyophilisation followed bysterilization in ethylene oxide.

Rama et al reported the cryopreservation of AM in 10% dimethyl sulfoxide(DMSO) instead of glycerol and achieved a cell viability of about 40%.

Two placental tissue graft products containing living cells, Apligrafand Dermagraft, are currently commercially available. Both Apligraf andDermagraft comprise ex vivo cultured cells. Neither Apligraf norDermagraft comprise stem cells. Furthermore, neither Apligraf norDermagraft comprise Insulin-like Growth Factor Binding Protein-1(IGFBP-1) and adiponectin, which are key factors in the natural woundhealing process. In addition, neither Apligraf nor Dermagraft exhibit aprotease-to-protease inhibitor ratio favorable for wound healing. Aswound healing is a multi-factorial biological process, many factors areneeded to properly treat a wound; products having non-native cellularpopulations are less capable of healing wounds relative to a producthaving an optimal population of cells representing the native array. Itwould represent an advance in the art to provide a chorion-derivedbiologic skin substitute comprising a population of cells representingthe native array of factors, including, for example, growth factors andcytokines.

Apligraf is a living, bi-layered skin substitute manufactured usingneonatal foreskin keratinocytes and fibroblasts with bovine Type Icollagen. As used in this application, Apligraf refers to the productavailable for commercial sale in November 2009.

Dermagraft is cryopreserved human fibroblasts derived from newbornforeskin tissue seeded on extracellular matrix. According to its productliterature, Dermagraft requires three washing steps before use whichlimits the practical implementation of Dermagraft as a wound dressingrelative to products that require less than three washing steps. As usedin this application, Dermagraft refers to the product available forcommercial sale in November 2009.

Engineered wound dressings such as Apligraf and Dermagraft do notprovide the best potential for wound healing because they comprisesub-optimal cellular compositions and therefore do not provide properwound healing. For example, neither Apligraf nor Dermagraft comprisesstem cells and, as a result, the ratio between different factorssecreted by cells does not enable efficient wound healing. Additionally,some factors that are important for wound healing, including EGF,IGFBP1, and adiponectin, are absent from both Apligraf and Dermagraft.Additionally, some factors, including MMPs and TIMPs, are present inproportions that differ greatly from the proportions found in thenatural wound healing process; this difference significantly alters,among other things, upstream inflammatory cytokine pathways which inturn allows for sub-optimal micro-environments at the wound site.

Paquet-Fifield et al. report that mesenchymal stem cells and fibroblastsare important for wound healing (J Clin Invest, 2009, 119: 2795). Noproduct has yet been described that comprise mesenchymal stem cells andfibroblasts.

Both MMPs and TIMPs are among the factors that are important for woundhealing. However, expression of these proteins must be highly regulatedand coordinated. Excess of MMPs versus TIMPs is a marker of poor chronicwound healing (Liu et al, Diabetes Care, 2009, 32: 117; Mwaura et al,Eur J Vasc Endovasc Surg, 2006, 31: 306; Trengove et al, Wound Rep Reg,1999, 7: 442; Vaalamo et al, Hum Pathol, 1999, 30: 795).

α2-macroglobulin is known as a plasma protein that inactivatesproteinases from all 4 mechanistic classes: serine proteinases, cysteineproteinases, aspartic proteinases, and metalloproteinases (Borth et al.,FASEB J, 1992, 6: 3345; Baker et al., J Cell Sci, 2002, 115:3719).Another important function of this protein is to serve as a reservoirfor cytokines and growth factors, examples of which include TGF, PDGF,and FGF (Asplin et al, Blood, 2001, 97: 3450; Huang et al, J Biol Chem,1988; 263: 1535). In chronic wounds like diabetic ulcers or venousulcers, the presence of high amount of proteases leads to rapiddegradation of growth factors and delays in wound healing. Thus, aplacental membrane wound dressing comprising α2-macroglobulin wouldconstitute an advance in the art.

An in vitro cell migration assay is important for assessing the woundhealing potential of a skin substitute. The process of wound healing ishighly complex and involves a series of structured events controlled bygrowth factors (Goldman, Adv Skin Wound Care, 2004, 1:24). These eventsinclude increased vascularization, infiltration by inflammatory immunecells, and increases in cell proliferation. The beginning stages ofwound healing revolve around the ability of individual cells to polarizetowards the wound and migrate into the wounded area in order to closethe wound area and rebuild the surrounding tissue. An assay capable ofevaluating the wound healing potential of skin substitutes by examiningthe correlation between cell migration and wound healing would representan advance in the art.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutically acceptable placentalproduct.

A placental product according to the present invention comprises animmunocompatible amniotic membrane in a cryopreservation medium(optionally cryopreserved) and viable native therapeutic cells andnative therapeutic factors.

In some embodiments, the amniotic membrane of the placental product isselectively devitalized.

There is now provided a placental product that is selectively depletedof substantially all immunogenic cells.

There is now provided a placental product that does not contain ex vivocultured cells.

In some embodiments, the placental product further comprises a chorionicmembrane that is selectively devitalized.

There is now provided a placental product that comprises at least one ofEpidermal Growth Factor, IGFBP1, and Adiponectin.

Optionally, the therapeutic factors include one or more of IGFBP1,adiponectin, α2-macroglobulin, bFGF, and EGF. Optionally, thetherapeutic factors include MMP-9 and TIMP1, wherein the ratio ofMMP-9:TIMP1 is from about 7 to about 10. Optionally, the therapeuticfactors include IGFBP1, adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9,and TIMP1. Optionally, the therapeutic factors include IGFBP1,adiponectin, α2-macroglobulin, bFGF, MMP-9, and TIMP1, wherein the ratioof MMP-9:TIMP1 is from about 7 to about 10 to one. Optionally, thetherapeutic factor is present in a substantial amount in comparison tounprocessed human placental membrane. Optionally, each placental productembodiment optionally is devoid of ex-vivo expanded cultured cells.

The present invention also provides a method of manufacturing aplacental product comprising: obtaining a placenta, wherein the placentacomprises an amniotic membrane, selectively depleted of immunogenicity,and cryopreserving the placenta, thereby providing a placental product.According to the present invention, the step of selective depletioncomprises removing immunogenic cells (e.g. CD14+ macrophages andvascularized tissue-derived cells) and/or immunogenic factors (e.g.TNFα). Optionally, the step of selective depletion comprises removingCD14+ macrophages by refrigerating the placental product for a period oftime (e.g. about 30-60 mins.) at a temperature above freezing (e.g. at2-8° C.), and then freezing, whereby CD14+ macrophages are selectivelykilled relative to therapeutic cells.

The present invention also provides a method of screening a placentalproduct for therapy comprising assaying the placental product forimmunogenicity and/or therapeutic value. Optionally, the step ofassaying the placental product for immunogenicity comprises a MixedLymphocyte Reaction (MLR) and/or Lipopolysaccharide (LPS)-induced TumorNecrosis Factor (TNF)-α secretion. Optionally, the step of assaying theplacental product for therapeutic value comprises assaying the placentalproduct for cell migration induction.

The present invention also provides a method of treating a subjectcomprising administering a placental product to the subject. Optionally,the step of administering comprises applying the placental product to awound, for example, topically applying the placental product to a skinwound. In one embodiment, a placental product is used in a tendon orligament surgery to promote healing of a tendon or ligament.

The present inventors have identified a need for the development ofamniotic membrane products comprising at least one of IGFBP1, andadiponectin, providing superior wound healing properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a-h depicts freezing rates of various freezing methods.

FIG. 2 depicts process cell recovery for amniotic membrane as a functionof cryo volume.

FIG. 3 depicts process cell recovery for chorionic membrane as afunction of cryo volume.

FIG. 4 depicts the effects of refrigeration time and freezing parameterson process (cryopreservation) cell recovery for the amniotic membrane.

FIG. 5 depicts the effects of refrigeration time and freezing parameterson process (cryopreservation) cell recovery for the chorionic membrane.

FIG. 6A-F shows representative images of the live/dead staining of theepithelial layer of fresh amniotic membrane.

FIG. 7 depicts expression of IL-2sR from T-cells stimulated by placentalderived cells from various membrane preparations.

FIG. 8 depicts expression of IL-2sR from T-cells stimulated by placentalderived cells from various membrane preparations after cryopreservation.

FIG. 9A-C depicts LPS stimulated TNF a released from various membranepreparations.

FIG. 10A-C shows images of cultured cells isolated from various membranepreparations.

FIG. 11 depicts a correlation between IL-2sR release in an MLR assay ofvarious membrane preparations and the number of CD45+ cells.

FIG. 12 depicts the IL-2sR release in an MLR assay of amniotic membranepreparations and the number of CD45+ cells.

FIG. 13A-C depicts expression of EGF (A), IGFBP1 (B), and Adiponectin(C) in amniotic and/or chorionic membranes.

FIG. 14 depicts expression of IFN-2α and TGF-β3 in amniotic membranehomogenates.

FIG. 15A-B depicts expression of BMP-2, BMP-4, PLAB, PIGF (A), and IGF-1(B) in amniotic membrane homogenates.

FIG. 16 depicts the ratio of MMPs to TIMPs in various membrane products.

FIG. 17 depicts expression of EGF in chorion and amnion membranesmeasured by ELISA in two separate placenta donors.

FIG. 18 depicts the Cell Biolabs 24-well Cytoselect wound healing assay.

FIG. 19 depicts representative images of HMVECs treated with 5%conditioned media from amniotic, chorionic, or a combination ofamniotic/chorionic preparations.

FIG. 20A-E depicts the remarkable efficacy of placental products fortreating diabetic foot ulcers in patient 1.

FIG. 21A-E depicts the remarkable efficacy of placental products fortreating diabetic foot ulcers in patient 2.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following definitions apply:

“Examplary” (or “e.g.” or “by example”) means a non-limiting example.

“hCMSCs: means human chorionic membrane stromal cells. hCMSCs aregenerally positive for CD73?, CD70, CD90, CD105, and CD166; negative forCD45 and CD34. hCMSCs differentiate to mesodermal lineages (osteogenic,chondrogenic, and adipogenic).

“Selective depletion of immunogenicity” or “selective depletion ofimmunogenic cells or factors” or “selective depletion” means a placentalproduct that retains live therapeutic cells and/or retains therapeuticefficacy for the treatment of tissue injury yet is free, substantiallyfree, or depleted of at least one of immune cell type (e.g. CD14+macrophages, trophoblasts, and/or vascular-tissue derived cells) and/orimmunogenic factor that are otherwise present in a native placenta oramniotic membrane.

“MSC” means mesenchymal stem cells and include fetal, neonatal, adult,or post-natal. “MSCs” include amniotic MSCs (AMSCs). MSCs generallyexpress one or more of CD73, CD90, CD105, and CD166.

“Native cells” means cells that are native, resident, or endogenous tothe placental membrane, i.e. cells that are not exogenously added to theplacental membrane.

“Native factors” means placental membrane factors that are native,resident, or endogenous to the placental membrane, i.e. factors that arenot exogenously added to the placental membrane.

“Placental products” means the instant placental products disclosed andclaimed herein.

“Substantial amount” of an element of the present invention, e.g. nativefactors, native cells, therapeutic factors, or selective depletion,means a value at least about 10% or more in comparison to anunprocessed, not cryopreserved, fresh membrane sample. A substantialamount can optionally be at least about 50%.

“Therapeutic cells” or “beneficial cells” means stromal cells, MSCs,fibroblasts, and/or epithelial cells.

“Therapeutic factors” means placenta- or amniotic membrane-derivedfactors that promote wound healing. Examples include IGFBP1,adiponectin, α2-macroglobulin, and/or bFGF. Other examples include MMP-9and TIMP1.

“Stromal cells” refers to a mixed population of cells present(optionally in native proportions) composed of neonatal mesenchymal stemcells and neonatal fibroblasts. Both neonatal mesenchymal stem cells andneonatal fibroblasts are immunoprivileged; neither express surfaceproteins present on immunogenic cell types.

In some embodiments, the present technology discloses placental productsfor clinical use, including use in wound healing such as diabetic footulcers, venous leg ulcers, and burns. The manufacturing processoptionally eliminates essentially all potentially immunogenic cells fromthe placental membrane while preserving of specific cells that play animportant role in wound healing.

In some embodiments, the present technology discloses a placentalproduct that is selectively devitalized. There is now provided aplacental product that is selectively depleted of substantially allimmunogenic cells. There is now provided a placental product that doesnot contain ex vivo cultured cells. There is now provided a placentalproduct that comprises at least one of IGFBP1, and adiponectin. There isnow provided a placental product that comprises Epidermal Growth Factor,IGFBP1. There is now provided a placental product that comprisesadiponectin.

In some embodiments, the present technology discloses a method ofcyropreserving a placental product that preserves the viability ofspecific beneficial cells that are the primary source of factors for thepromotion of healing to the wound healing process while selectivelydepleting immunogenic cells (e.g. killing or rendering non-immunogenic).

In some embodiments, the present technology discloses a bioassay to testimmunogenicity of manufactured placental products.

In some embodiments, the present technology discloses a placentalproduct exhibiting a therapeutic ratio of MMP to TIMP comparable to thatexhibited in vivo. The present inventors have identified a need for thedevelopment of placental products exhibiting a ratio of MMP-9 and TIMP1of about 7-10 to one.

In some embodiments, the present technology discloses a placentalproduct that comprises α2-macroglobulin.

There is now provided a placental product that inactivates substantiallyall serine proteinases, cysteine proteinases, aspartic proteinases, andmetalloproteinases present in the amniotic membrane. There is nowprovided a placental product that inactivates substantially all serineproteinases present in the amniotic membrane. There is now provided aplacental product that inactivates substantially all cysteineproteinases present in the amniotic membrane. There is now provided aplacental product that inactivates substantially all asparticproteinases present in the amniotic membrane. There is now provided aplacental product that inactivates substantially all metalloproteinasespresent in the amniotic membrane.

In some embodiments, the present technology discloses a placentalproduct that comprises bFGF.

In some embodiments, the present technology discloses a placentalproduct exhibiting a protease-to-protease inhibitor ratio favorable forwound healing.

In some embodiments, the present technology discloses a cell migrationassay capable of evaluating the wound-healing potential of a placentalproduct.

IGFBP1 and adiponectin are among the factors that are important forwound healing. Evaluation of proteins derived from placental productsprepared according to the presently disclosed technology reveal that EGFis one of the major factors secreted in higher quantities by thesetissues. Additionally, the importance of EGF for wound healing togetherwith high levels of EGF detected in the presently disclosed amnioticmembranes support selection of EGF as a potency marker for evaluation ofmembrane products manufactured for clinical use pursuant to the presentdisclosure.

The present technology discloses a cryopreservation procedure for aplacental product that selectively depletes immunogenic cells andpreserves the viability of other beneficial cells (including at leastone or 2 or all of mesenchymal stem cells, epithelial cells andfibroblasts. In some embodiments, the beneficial cells are the primarysource of factors for the promotion of healing.

Placental products, their usefulness, and their immunocompatability aresurprisingly enhanced by depletion of maternal trophoblast and selectiveelimination of CD14+ fetal macrophages. Immunocompatability can bedemonstrated by any means commonly known by the skilled artisan, suchdemonstration can be performed by the mixed Lymphocyte Reaction (MLR)and by lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-αsecretion.

The instant placental products contain bFGF optionally at a substantialconcentration.

The instant placental products optionally secrete factors that stimulatecell migration and/or wound healing. The presence of such factors can bedemonstrated by any commonly recognized method,

For example, commercially available wound healing assays exist (CellBiolabs) and cell migration can be assessed by cell line HMVEC (LonzaInc.). In one embodiment, conditioned medium from the present placentalproducts enhance cell migration.

The placental products disclosed herein are useful in treating a numberof wounds including: tendon repair, cartilage repair (e.g. femoralcondyle, tibial plateau), ACL replacement at the tunnel/bone interface,dental tissue augmentation, fistulas (e.g. Crohn's disease, G-tube,tracheoesophogeal), missing tissue at adhesion barriers (e.g. nasalseptum repair, vaginal wall repair, abdominal wall repair, tumorresection), dermal wounds (e.g. partial thickness burns, toxic epidermalnecrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g.diabetic ulcers (e.g. foot), venous leg ulcers), surgical wounds, herniarepair, tendon repair, bladder repair, periosteum replacement, keloids,organ lacerations, epithelial defects, and repair or replacement of atympanic membrane.

The placental products disclosed herein exhibit one or more of thefollowing properties beneficial to the wound healing process:

-   a. an epithelial cell layer, wherein the approximate number of cells    per cm2 of the amniotic membrane is about 10,000 to about 360,000 or    about 40,000 to about 90,000.-   b. a thick basement membrane (comprising one or more of Collagen    Type I, III, IV, laminin, and fibronectin),-   c. a stromal cell layer;-   d. an amniotic membrane with a thickness of about 20 to about 50 μm,-   e. high thrombin activity,-   f. low immunogenicity,-   g. cryopreserved/cryopreserveable,-   h. amniotic MSC,-   i. analgesic effect-   j. reduces scarring,-   k. anti-inflammatory proteins such as IL-1a and IL-10,-   l. anti-inflammatory suppression of lymphocyte reactivity in vitro,    for example by inhibition of CD8+ and CD4+ proliferation or    increased CD4+ Treg cells,-   m. antibacterial proteins such as defensins and allantoin    (bacteriolytic proteins),-   n. angiogenic and mitogenic factors that promote    re-epithelialization such as EGF, HGF, and VEGF,-   o. cells that are positive for CD70, CD90, CD105, and CD166 and    negative for CD45 and CD34,-   p. cells that express HLA-G,-   q. cells that express a placenta specific MHC-1 antigen important to    immune tolerance that inhibits both NK cytolysis and T-cell-mediated    cytolysis and activation of immune cells (e.g. interferon-γ    secretion),-   r. cells that express IDO and FAS ligand, which likely contribute to    immune tolerance,-   s. cells with a capacity to differentiate into 1—Human Amniotic    Epithelial Cells (hAECs)-   t. cells with a capacity to differentiate to neural, hepatocyte, and    pancreatic cells,-   u. Human Amniotic Membrane Stromal Cells (hAMSCs) differentiate to    mesodermal lineages (osteogenic, chondrogenic, and adipogenic) and    to all three germ layers-ectoderm (neural), mesoderm (skeletal    muscle, cardiomyocytic, and endothelial), and ectoderm (pancreatic),-   v. Cells that expression CD49d by hAMSCs distinguishes hAMSCs from    hAECs,-   w. hAMSCs that positive for the embryonic cytoplasmic marker Oct-4    that plays a role in maintaining pluripotency and self renewal,-   x. hAECs that are positive for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,    and negative for SSEA-4 and non-tumorogenic.

The present inventors have now identified a need for the development ofplacental products that do not contain ex vivo cultured cells.

The present inventors have now identified a need for the development ofplacental products comprising IGFBP1.

The present inventors have now identified a need for the development ofplacental products comprising adiponectin.

The present inventors have now identified a need for the development ofplacental products exhibiting a protease-to-protease inhibitor ratiofavorable for wound healing.

The present inventors have now identified a need for the development ofa method of cyropreserving placental products that preserves theviability of specific cells that are other beneficial cells that are theprimary source of factors for the promotion of healing to the woundhealing process while selectively depleting immunogenic cells fromchorionic membranes.

The present inventors have now identified a need for the development ofa bioassay to test immunogenicity of manufactured placental products.

The present inventors have now identified a need for the development ofplacental products exhibiting a ratio of MMP to TIMP comparable to thatexhibited in vivo. The present inventors have now identified a need forthe development of placental products exhibiting a ratio of MMP-9 andTIMP1 of about 7-10 to one.

The present inventors have now identified a need for the development ofplacental products that comprise α2-macroglobulin.

The present inventors have now identified a need for the development ofplacental products that inactivate serine proteinases, cysteineproteinases, aspartic proteinases, and metalloproteinases. The presentinventors have now identified a need for the development of placentalproducts that inactivate serine proteinases. The present inventors havenow identified a need for the development of placental products thatinactivate cysteine proteinases. The present inventors have nowidentified a need for the development of placental products thatinactivate aspartic proteinases. The present inventors have nowidentified a need for the development of placental products thatinactivate metalloproteinases.

The present inventors have now identified a need for the development ofplacental products that comprise bFGF.

The present inventors have now identified a need for the development ofa cell migration assay to evaluate the potential of placental membraneproducts.

The present inventors have now identified a need for the development ofa placental product for wound healing that comprises mesenchymal stemcells, epithelial cells and fibroblasts.

Placental Product

Overview

One embodiment of the present invention provides a placental productcomprising a cryopreservation medium and an amniotic membrane, whereinthe amniotic membrane comprises viable native therapeutic cells andnative therapeutic factors, and wherein the cryopreservation mediumcomprises a cryopreserving amount of a cryopreservative. According tothis embodiment, the amniotic membrane is substantially free of at leastone at least one or both of the following immunogenic cell types: CD14+macrophages and vascularized tissue-derived cells.

In one embodiment, the amniotic membrane comprises one or more layerswhich exhibit the architecture of the native amniotic membrane (e.g. hasnot been homogenized or treated with collagenase).

In one embodiment, the placental product is suitable for dermalapplication to a wound.

With the teachings provided herein, the skilled artisan can now producethe present placental products. The present disclosure provides methodsof manufacture that produce the technical features of the presentplacental products. Accordingly, in one embodiment, the placentalproduct is manufactured by steps taught herein. The present placentalproducts are not limited to products manufactured by the methods taughthere. For example, products of the present invention could be producedthrough methods that rely on screening steps; e.g. steps to screen forpreparations with the described technical features and superiorproperties.

The present placental product comprises one or more of the followingtechnical features:

-   a. the viable therapeutic native cells are capable of    differentiating into cells of more than one lineage (e.g.    osteogenic, adipogenic and/or chonodrogenic lineages),-   b. the native therapeutic factors include IGFBP1,-   c. the native therapeutic factors include adiponectin,-   d. the native therapeutic factors include α2-macroglobulin,-   e. the native therapeutic factors include bFGF,-   f. the native therapeutic factors include EGF,-   g. the native therapeutic factors include MMP-9 and TIMP1,-   h. the native therapeutic factors include MMP-9 and TIMP1 in a ratio    of about 7 to about 10,-   i. the placental product does not comprise ex-vivo cultured cells,-   j. the cryopreservative medium is present in an amount of greater    than about 20 ml or greater than about 50 ml,-   k. the cryopreservative comprises DMSO,-   l. cryopreservative comprises DMSO in a majority amount,-   m. the cryopreservation medium further comprises albumin, optionally    wherein the albumin is HSA,-   n. the cryopreservative comprises DMSO and albumin (e.g. HSA),-   o. comprises about 5,000 to about 240,000 cells/cm² or about 20,000    to about 60,000,-   p. the amniotic membrane comprises at least: about 2,000, or about    2,400, or about 4,000′ or about 6,000, or about 8,000, or about    10,000, or about 10,585, or about 15,000 stromal cells per unit cm²    of the amniotic membrane,-   q. the amniotic membrane comprises about 2,000 to about 15,000 of    stromal cells per cm² of the amniotic membrane,-   r. comprises stromal cells wherein at least: about 40%, or about    50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or    about 90%, or about 92.5% of the stromal cells are viable after a    freeze-thaw cycle,-   s. comprises stromal cells wherein about 40% to about 92.5% of the    stromal cells are viable after a freeze-thaw cycle,-   t. the amniotic membrane has a thickness of about 20 μm to about 50    μm,-   u. secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280    pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm    piece of the tissue product in a tissue culture medium and exposing    the tissue product to a bacterial lipopolysaccharide for about 20 to    about 24 hours,-   v. cryopreservation and thawing, secretes less than about any of:    420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue culture    medium upon placing a 2 cm×2 cm piece of the tissue product in a    tissue culture medium and exposing the tissue product to a bacterial    lipopolysaccharide for about 20 to about 24 hours,-   w. after refrigeration, cryopreservation and thawing, secretes less    than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a    tissue culture medium upon placing a 2 cm×2 cm piece of the tissue    product in a tissue culture medium and exposing the tissue product    to a bacterial lipopolysaccharide for about 20 to about 24 hours,-   x. further comprises an chorionic membrane,-   y. amniotic membrane comprises a layer of amniotic epithelial cells,-   z. further comprises an chorionic membrane, wherein the amniotic    membrane and the chorionic membrane are associated to one another in    the native configuration,-   aa. further comprises an chorionic membrane, wherein the amniotic    membrane and the chorionic membrane are not attached to one another    in the native configuration,-   bb. further comprises a chorionic membrane wherein the chorionic    membrane comprises about 2 to about 4 times more stromal cells    relative to the amniotic membrane,-   cc. does not comprise a chorionic membrane;-   dd. comprises chorionic membrane, wherein the chorionic membrane    comprises about 2 to about 4 times more stromal cells relative to    the amniotic membrane, and-   ee. is suitable for dermal application to a wound;    Cells

According to the present invention, a placental product comprises nativetherapeutic cells of the amniotic membrane. The cells comprise one ormore of stromal cells, MSCs, fibroblasts, and epithelial cells.

In one embodiment, the native therapeutic cells comprise viable stromalcells.

In one embodiment, the native therapeutic cells comprise viable MSCs.

In one embodiment, the native therapeutic cells comprise viablefibroblasts.

In one embodiment, the native therapeutic cells comprise viableepithelial cells.

In one embodiment, the native therapeutic cells comprise viable MSCs andviable fibroblasts.

In one embodiment, the native therapeutic cells comprise viable MSCs,viable fibroblasts, and viable epithelial cells.

In one embodiment, the native therapeutic cells comprise viable stromalcells and viable epithelial cells.

In one embodiment, the therapeutic native cells are viable cells capableof differentiating into cells of more than one lineage (e.g. osteogenic,adipogenic and/or chonodrogenic lineages).

In one embodiment, the placental product comprises about 10,000 to about360,000 or about 40,000 to about 90,000 cells per cm².

In one embodiment, the placental product comprises at least: about2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000,or about 10,000, or about 10,585, or about 15,000 stromal cells per unitcm² of the placental product.

In one embodiment, the placental product comprises about 2,000 to about15,000 of stromal cells per cm² of the placental product.

In one embodiment, the placental product comprises stromal cells whereinat least: about 40%, or about 50%, or about 60%, or about 70%, or about74.3%, or about 83.4 or about 90%, or about 92.5% of the stromal cellsare viable after a freeze-thaw cycle.

In one embodiment, the placental product comprises stromal cells whereinabout 40% to about 92.5% of the stromal cells are viable after afreeze-thaw cycle.

In one embodiment, the placental product comprises less than about 1% ofCD14+ macrophages per total cells.

In one embodiment, the amniotic membrane of the placental productcomprises about 2 to about 4 times less stromal cells relative to achorionic membrane of the same area derived from the same placenta.

In one embodiment, the placental product further comprises membranechorionic membrane containing about 2 to about 4 times more stromalcells relative to the amniotic membrane.

In one embodiment, the amniotic membrane of the placental productcomprises MSCs in an amount of: at least about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, about 1% to about10%, or about 3% to about 10%, relative to the total number of cells inthe amniotic membrane of the placental product. Optionally, at least:about 40%, about 50%, about 60%, or about 70% of the MSCs are viableafter a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental productcomprises fibroblasts in an amount of: about 1%, about 20%, about 5% toabout 15%, at least about 1%, at least about 2%, at least about 3%, orat least about 4 relative to the total number of cells in the amnioticmembrane of the placental product. Optionally, at least: about 40%,about 50%, about 60%, or about 70% of the fibroblasts are viable after afreeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental productcomprises stromal cells in an amount of: about 5% to about 40%, about 5%to about 30%, about 10% to about 30%, about 15% to about 25%, at leastabout 5%, at least about 10%, or at least about 15%, relative to thetotal number of cells in the amniotic membrane of the placental product.Optionally, at least: about 40%, about 50%, about 60%, or about 70% ofthe stromal cells are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental productcomprises epithelial cells in an amount of: about 60% to about 90%,about 70% to about 90%, about 40% to about 90%, about 50% to about 90%,at least about 40%, at least about 50%, least about 60%, or at leastabout 70%, relative to the total number of cells in the amnioticmembrane of the placental product. Optionally, at least: about 40%,about 50%, about 60%, or about 70% of the epithelial cells are viableafter a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental productcomprises MSCs and functional macrophages in a ratio of greater thanabout any of: 5:1, 7:1, or 10:1.

In one embodiment, the amniotic membrane of the placental productcomprises fibroblasts and functional macrophages in a ratio of greaterthan about any of: 10:1, 15:1, 20:1, or 25:1.

In one embodiment, the amniotic membrane of the placental productcomprises fibroblasts and MSCs in a ratio of: about 4:1 to about 1:1 orabout 3:1 to about 3:2, or about 2:1.

In one embodiment, the amniotic membrane of the placental productcomprises MSCs in an amount of: at least about 1,000 cells/cm², at leastabout 2,000 cells/cm², about 1,000 to about 5,000 cells/cm², or about2,000 to about 5,000 cells/cm². Optionally, at least: about 40%, about50%, about 60%, or about 70% of the MSCs are viable after a freeze-thawcycle.

In one embodiment, the amniotic membrane of the placental productcomprises fibroblasts in an amount of: at least about 2,000 cells/cm²,at least about 4,000 cells/cm², about 2,000 to about 9,000 cells/cm², orabout 2,000 to about 9,000 cells/cm². Optionally, at least: about 40%,about 50%, about 60%, or about 70% of the fibroblasts are viable after afreeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental productcomprises stromal cells in an amount of: at least about 4,000, at leastabout 8,000 cells/cm², about 4,000 to about 18,000 cells/cm², or about4,000 to about 18,000 cells/cm². Optionally, at least: about 40%, about50%, about 60%, or about 70% of the stromal cells are viable after afreeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental productcomprises epithelial cells in an amount of: at least about 10,000cells/cm², at least about 20,000 cells/cm², at least about 32,000cells/cm², about 10,000 to about 72,000 cells/cm², about 20,000 to about72,000 cells/cm², or about 32,000 to about 72,000 cells/cm² Optionally,at least: about 40%, about 50%, about 60%, or about 70% of theepithelial cells are viable after a freeze-thaw cycle.

In one embodiment, the amniotic membrane of the placental productcomprises functional macrophages in an amount of: less than about 3,000cells/cm², less than about 1,000 cells/cm², or less than about 500cells/cm².

In one embodiment, the placental product comprises a layer of amnioticepithelial cells.

In one embodiment, the placental product comprises a chorionic membranebut is substantially free of trophoblasts.

In one embodiment, the placental product is substantially free offunctional CD14+ macrophages.

In one embodiment, the placental product is substantially free ofvascularized tissue-derived cells.

In one embodiment, the placental product is substantially free oftrophoblasts, functional CD14+ macrophages, and vascularizedtissue-derived cells. Optionally, the placental product comprises viablestromal cells. Optionally, the placental product comprises viable MSCs.Optionally, the placental product comprises viable fibroblasts.Optionally, the placental product comprises viable epithelial cells.Optionally, the placental product comprises viable MSCs, fibroblasts,and epithelial cells.

In one embodiment, the placental product comprises a chorionic membranebut is substantially free of maternal decidual cells.

In one embodiment, the placental product comprises a chorionic membranebut is substantially free of maternal leukocytes and/or trophoblastcells.

In one embodiment, the placental product is substantially free ofex-vivo cultured cells.

Placental Factors

According to the present invention, a placental product comprises nativetherapeutic factors of the amniotic membrane.

In one embodiment, the factors include one or more of: IGFBP1,adiponectin, α2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally,the factors are present in amounts/cm² that are substantially similar tothat of a native amniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include IGFBP1, adiponectin,α2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally, the factorsare present in ratios that are substantially similar to that of a nativeamniotic membrane or layer thereof. Optionally, the factors are presentin amounts/cm² that are substantially similar to that of a nativeamniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include MMP-9 and TIMP1 in a ratio ofabout 7 to about 10 (e.g about 7). Optionally, the factors are presentin amounts/cm² that are substantially similar to that of a nativeamniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the factors include one or more (e.g. a majority orall) of the factors listed in Table 16. Optionally, the factors arepresent in ratios that are substantially similar to that of a nativeamniotic membrane or layer thereof. Optionally, the factors are presentin amounts/cm² that are substantially similar to that of a nativeamniotic membrane or layer thereof (e.g. ±10% or 20%).

In one embodiment, the placental product or layer thereof comprisessubstantially less TNF-α/cm² than a native amniotic membrane or layerthereof, respectively.

In one embodiment, the placental product or layer thereof secretessubstantially less TNF-α/cm² than a native placental product or layerthereof, respectively.

In one embodiment, the placental product secretes less than about anyof: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-α into a tissue culturemedium upon placing a 2 cm×2 cm piece of the tissue product in a tissueculture medium and exposing the tissue product to a bacteriallipopolysaccharide for about 20 to about 24 hours.

In one embodiment, after cryopreservation and thawing, the placentalproduct secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280pg/mL TNF-α into a tissue culture medium upon placing a 2 cm×2 cm pieceof the tissue product in a tissue culture medium and exposing the tissueproduct to a bacterial lipopolysaccharide for about 20 to about 24hours;

In one embodiment, after refrigeration, cryopreservation and thawing,the placental product secretes less than about any of: 420 pg/mL, 350pg/mL, or 280 pg/mL TNF-α into a tissue culture medium upon placing a 2cm×2 cm piece of the tissue product in a tissue culture medium andexposing the tissue product to a bacterial lipopolysaccharide for about20 to about 24 hours.

In one embodiment, the placental product further comprises anexogenously added inhibitor of TNF-α. Optionally, the inhibitor of TNF-αis IL-10.

In one embodiment, the product has been treated with an antibiotic

Architecture

A placental product of the present invention comprises one or morelayers which exhibit the architecture of the native amniotic membrane.With the teachings provided herein, the skilled artisan will recognizeplacental layers that exhibit native architecture, for example, layersthat have not been homogenized or treated with collagenase or otherenzyme that substantially disrupts the layer.

In one embodiment, the placental product comprises a stromal cell layerwith native architecture of the amniotic membrane.

In one embodiment, the placental product comprises a basement membranewith native architecture of the amniotic membrane.

In one embodiment, the placental product comprises an epithelial celllayer with native architecture of the amniotic membrane.

In one embodiment, the placental product comprises a stromal cell layerand a basement layer with native architecture of the amniotic membrane.

In one embodiment, the placental product comprises a stromal layer, abasement layer, and an epithelial cell layer with native architecture ofthe amniotic membrane.

In one embodiment, the placental product or amniotic membrane thereofhas a thickness of about 20 μm to about 50 μm.

In one embodiment, the placental product comprises a chorionic membranebut is substantially free of trophoblasts. In one embodiment, theplacental product comprises a basement membrane with native architectureof the chorionic membrane and the chorionic membrane is substantiallyfree of trophoblasts. Optionally, the maternal side of the chorionicmembrane comprises fragments of extracellular matrix proteins in aconcentration substantially greater than that of a native chorionicmembrane. Optionally, the placental product has been treated withDispase (e.g. Dispase II) and/or a substantial portion of theextracellular matrix protein fragments comprises terminal leucine orphenylalanine.

In one embodiment, the placental product further comprises a chorionicmembrane. Optionally, the amniotic membrane and the chorionic membranein the placental product are associated to one another in the nativeconfiguration. Alternatively, the amniotic membrane and the chorionicmembrane are not attached to one another in the native configuration.

In one embodiment, the placental product does not comprise a chorionicmembrane.

Formulation

According to the present invention, the placental product can beformulated with a cryopreservation medium.

In one embodiment, the cryopreservation medium comprising one or morecell-permeating cryopreservatives, one or more non cell-permeatingcryopreservatives, or a combination thereof.

Optionally, the cryopreservation medium comprises one or morecell-permeating cryopreservatives selected from DMSO, a glycerol, aglycol, a propylene glycol, an ethylene glycol, or a combinationthereof.

Optionally, the cryopreservation medium comprises one or more noncell-permeating cryopreservatives selected from polyvinylpyrrolidone, ahydroxyethyl starch, a polysacharide, a monosaccharides, a sugaralcohol, an alginate, a trehalose, a raffinose, a dextran, or acombination thereof.

Other examples of useful cryopreservatives are described in“Cryopreservation” (BioFiles Volume 5 Number 4—Sigma-Aldrich®datasheet).

In one embodiment, the cryopreservation medium comprises acell-permeating cryopreservative, wherein the majority of thecell-permeating cryopreservative is DMSO. Optionally, thecryopreservation medium does not comprise a substantial amount ofglycerol.

In one embodiment, the cryopreservation medium comprises DMSO.Optionally, the cryopreservation medium does not comprise glycerol in amajority amount. Optionally, the cryopreservation medium does notcomprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises additionalcomponents such as albumin (e.g. HSA or BSA), an electrolyte solution(e.g. Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 15%albumin by weight and about 5% to about 20% cryopreservative by volume(e.g. about 10%). Optionally, the cryopreservative comprises DMSO (e.g.in a majority amount).

In one embodiment, the placental product is formulated in greater thanabout 20 ml or greater than about 50 ml of cryopreservation medium.Optionally, the cryopreservative comprises DMSO (e.g. in a majorityamount). Optionally, the cryopreservation medium does not comprise asubstantial amount of glycerol.

In one embodiment, the placental product is placed on nitrocellulosepaper.

In one embodiment, the placenta is cut into a plurality of sections.Optionally, the sections are less than about 10 cm×10 cm. Optionally,the sections are between about 2 cm×2 cm and 5 cm×5 cm.

Manufacture

Overview

A placental product of the present invention can manufactured from aplacenta in any suitable manner that provides the technical featurestaught herein. According to the present invention, a placenta productcomprises at least an immunocompatible amniotic membrane.

In one embodiment, a placental product is manufactured by a methodcomprising:

-   a. obtaining a placenta,-   b. selectively depleting the placenta of immunogenicity; and-   c. cryopreserving the placenta.

Optionally, the method comprises a step of removing vascularized tissuefrom the placenta, for example, by lysing red blood cells, by removingblood clots, or a combination thereof.

Optionally, the method comprises a step of treating the placenta withone or more antibiotics.

Optionally, the method comprises a step of selectively depleting CD14+macrophages, optionally as demonstrated by a substantial decrease in LPSstimulation of TNFα release.

Optionally, the step of cryopreserving the placenta comprises freezingthe placenta in a cryopreservation medium which comprises one or morecell-permeating cryopreservatives, one or more non cell-permeatingcryopreservatives, or a combination thereof.

Optionally, the step of cryopreserving the placenta comprisesrefrigerating for a period of time and then freezing, therebyselectively depleting CD14+ macrophages optionally as demonstrated by asubstantial decrease in LPS stimulation of TNFα release.

Optionally, the method comprises retaining a layer of epithelial cellsof the amniotic membrane.

Optionally, the method comprises a step of removing the chorionicmembrane or portion thereof. Optionally, the method comprises removingtrophoblasts from the chorionic membrane while retaining the stromalcell layer, reticular layer, and/or basement membrane of the chorionicmembrane.

An exemplary placental product of the present invention can bemanufactured or provided with a bandage or wound dressing.

Immunocompatability and Selective Depletion

In one embodiment, the invention the placental product isimmunocompatible. Immunocompatability can be accomplished by anyselective depletion step that removes immunogenic cells or factors orimmunogenicity from the placenta (or amniotic membrane thereof).

In one embodiment, the placental product is made immunocompatible byselectively depleting it of functional immunogenic cells. A placenta canbe made immunocompatible by selectively removing immunogenic cells fromthe placenta (or amniotic membrane thereof) relative to therapeuticcells. For example, immunogenic cells can be removed by killing theimmunogenic cells or by purification of the placenta there from.

In one embodiment, the placental product is made immunocompatible byselectively depleting trophoblasts, for example, by removal of thetrophoblast layer.

In one embodiment, the placenta is made immunocompatible by selectivedepletion of functional CD14+ macrophages, optionally as demonstrated bya substantial decrease in LPS stimulation of TNFα release or by MLRassay.

In one embodiment, the placenta is made immunocompatible by selectivedepletion of vascularized tissue-derived cells.

In one embodiment, the placenta is made immunocompatible by selectivedepletion of functional CD14+ macrophages, trophoblasts, andvascularized tissue-derived cells.

In one embodiment, the placenta product is made immunocompatible byselective depletion of trophoblasts and/or CD14+ macrophages, optionallyas demonstrated by a substantial decrease in LPS stimulation of TNFαrelease or by MLR assay.

Trophoblast Removal

In one embodiment, Immunocompatability (or selective depletion) isaccomplished by removal or depletion of trophoblasts from the placentalproduct. Trophoblasts can be removed by removing the chorionic membranefrom the placental product or by removing trophoblasts from thechorionic membrane while retaining at least one of the basement layer,reticular layer, or stromal cell layer of the chorionic membrane.Surprisingly, such a placental product has one or more of the followingsuperior features:

-   a. is substantially non-immunogenic;-   b. provides remarkable healing time; and-   c. provides enhanced therapeutic efficacy.

In one embodiment, trophoblasts are removed while retaining the basementlayer, reticular layer, and/or stromal cell layer of the chorionicmembrane.

Trophoblasts can be removed in any suitable manner which substantiallydiminishes the trophoblast content of the placental product. Optionally,the trophoblasts are selectively removed or otherwise removed withouteliminating a substantial portion of one or more therapeutic componentsfrom the chorionic membrane (e.g. MSCs, placental factors, etc).Optionally, a majority (e.g. substantially all) of the trophoblasts areremoved.

One method of removing trophoblasts comprises treating the placenta(e.g. chorion or amnio-chorion) with a digestive enzyme such as dispase(e.g. dispase II) and separating the trophoblasts from the placenta.Optionally, the step of separating comprises mechanical separation suchas peeling or scraping. Optionally, scraping comprises scraping with asoft instrument such as a finger.

One method of removing trophoblasts comprises treating the chorionicmembrane with dispase for about 30 to about 45 minutes separating thetrophoblasts from the placenta. Optionally, the dispase is provided in asolution of about less than about 1% (e.g. about 0.5%). Optionally, thestep of separating comprises mechanical separation such as peeling orscraping. Optionally, scraping comprises scraping with a soft instrumentsuch as a finger.

Useful methods of removing trophoblasts from a placenta (e.g. chorion)are described by Portmann-Lanz et al. (“Placental mesenchymal stem cellsas potential autologous graft for pre- and perinatal neuroregeneration”;American Journal of Obstetrics and Gynecology (2006) 194, 664-73),(“Isolation and characterization of mesenchymal cells from human fetalmembranes”; Journal Of Tissue Engineering And Regenerative Medicine2007; 1: 296-305), and (Concise Review: Isolation and Characterizationof Cells from Human Term Placenta: Outcome of the First InternationalWorkshop on Placenta Derived Stem Cells”).

In one embodiment, trophoblasts are removed before cryopreservation.

Macrophage Removal

In one embodiment, functional macrophages are depleted or removed fromthe placental product. Surprisingly, such a placental product has one ormore of the following superior features:

-   a. is substantially non-immunogenic;-   b. provides remarkable healing time; and-   c. provides enhanced therapeutic efficacy.

Functional macrophages can be removed in any suitable manner whichsubstantially diminishes the macrophage content of the placentalproduct. Optionally, the macrophages are selectively removed orotherwise removed without eliminating a substantial portion of one ormore therapeutic components from the placenta (e.g. MSCs, placentalfactors, etc). Optionally, a majority (e.g. substantially all) of themacrophages are removed.

One method of removing immune cells such as macrophages compriseskilling the immune cells by rapid freezing rates such as 60-100° C./min.

Although immune cells can be eliminated by rapid freezing rates, such amethod can also be detrimental to therapeutic cells such as stromalcells (e.g. MSCs). The present inventors have discovered a method ofselectively killing CD14+ macrophages can be selectively killed byrefrigerating the placenta for a period of time (e.g. for at least about10 min such as for about 30-60 mins) at a temperature above freezing(e.g. incubating at 2-8° C.) and then freezing the placenta (e.g.incubating at −80° C.±5° C.). Optionally, the step of freezing comprisesfreezing at a rate of less than 10°/min (e.g. less than about 5°/minsuch as at about 1°/min).

In one embodiment, the step of refrigerating comprises soaking theplacenta in a cryopreservation medium (e.g. containing DMSO) for aperiod of time sufficient to allow the cryopreservation medium topenetrate (e.g. equilibrate with) the placental tissues. Optionally, thestep of freezing comprises reducing the temperature at a rate of about1°/min. Optionally, the step of freezing comprises freezing at a rate ofless than 10°/min (e.g. less than about 5°/min such as at about 1°/min).

In one embodiment, the step of refrigerating comprises soaking theplacenta in a cryopreservation medium (e.g. containing DMSO) at atemperature of about −10-15° C. (e.g. at 2-8° C.) for at least about anyof: 10 min, 20 min, 30 min, 40 min, or 50 min. In another embodiment thestep of refrigerating comprises soaking the placenta in acryopreservation medium (e.g. containing DMSO) at a temperature of about−10-15° C. (e.g. at 2-8° C.) for about any of: 10-120, 20-90 min, or30-60 min. Optionally, the step of freezing comprises freezing at a rateof less than 10°/min (e.g. less than about 5°/min such as at about1°/min).

Removal of Vascularized Tissue-Derived Cells

In one embodiment, vascularized tissue-derived cells (or vascularizedtissue) are depleted or removed from the placental product.Surprisingly, such a placental product has one or more of the followingsuperior features:

-   a. is substantially non-immunogenic;-   b. provides remarkable healing time; and-   c. provides enhanced therapeutic efficacy.

Vascularized tissue-derived cells can be removed in any suitable mannerwhich substantially diminishes such cell content of the placentalproduct. Optionally, the vascularized tissue-derived cells areselectively removed or otherwise removed without eliminating asubstantial portion of one or more therapeutic components from theplacenta (e.g. MSCs, placental factors, etc).

In one embodiment, removal of vascularized tissue-derived cellscomprises rinsing the amniotic membrane (e.g. with buffer such as PBS)to remove gross blood clots and any excess blood cells.

In one embodiment, removal of vascularized tissue-derived cellscomprises treating the amniotic membrane with an anticoagulant (e.g.citrate dextrose solution).

In one embodiment, removal of vascularized tissue-derived cellscomprises rinsing the amniotic membrane (e.g. with buffer such as PBS)to remove gross blood clots and any excess blood cells, and treating theamniotic membrane with an anticoagulant (e.g. citrate dextrosesolution).

In one embodiment, the chorionic membrane is retained and removal ofvascularized tissue-derived cells comprises separating the chorion fromthe placenta by cutting around the placental skirt on the side oppositeof the umbilical cord. The chorion on the umbilical side of the placentais not removed due to the vascularization on this side.

In one embodiment, the chorionic membrane is retained and removal ofvascularized tissue-derived cells comprises separating the chorion fromthe placenta by cutting around the placental skirt on the side oppositeof the umbilical cord and rinsing the amniotic membrane and chorionicmembrane (e.g. with buffer such as PBS) to remove gross blood clots andany excess blood cells.

In one embodiment, the chorionic membrane is retained and removal ofvascularized tissue-derived cells comprises separating the chorion fromthe placenta by cutting around the placental skirt on the side oppositeof the umbilical cord and treating the amniotic membrane and chorioncmembrane with an anticoagulant (e.g. citrate dextrose solution).

In one embodiment, the chorionic membrane is retained and emoval ofvascularized tissue-derived cells comprises separating the chorion fromthe placenta by cutting around the placental skirt on the side oppositeof the umbilical cord, rinsing the chorionic membrane amniotic membrane(e.g. with buffer such as PBS) to remove gross blood clots and anyexcess blood cells, and treating the amniotic membrane with ananticoagulant (e.g. citrate dextrose solution).

Selective Depletion of Immunogenicity as Demonstrated by a SubstantialDecrease in LPS Stimulation of TNFα Release.

In one embodiment, the placental product is selectively depleted ofimmunogenicity as demonstrated by a reduction in LPS stimulated TNF-αrelease. In one embodiment, the placental product is selectivelydepleted of macrophages.

In one embodiment, TNF-α is depleted by killing or removal ofmacrophages.

In one embodiment, TNF-α is functionally depleted by treatment withIL-10, which suppresses TNF-α secretion.

Preservation

A placental product of the present invention may be used fresh or may bepreserved for a period of time. Surprisingly, cryopreservation resultsin immunocompatible placental products.

In one embodiment, a placental product is cryopreserved. A placentalproduct may be cryopreserved by incubation at freezing temperatures(e.g. a −80° C.±5° C.) in a cryopreservative medium.

Cryopreservation can comprise, for example, incubating the placentalproduct at 4° C. for 30-60 min, and then incubating at −80° C. untiluse. The placental product may then be thawed for use. Optionally, theplacental product is cryopreserved in a manner such that cell viabilityis retained surprisingly well after a freeze-thaw cycle.

In one embodiment, cryopreservation comprises storage in acryopreservation medium comprising one or more cell-permeatingcryopreservatives, one or more non cell-permeating cryopreservatives, ora combination thereof. Optionally, the cryopreservation medium comprisesone or more cell-permeating cryopreservatives selected from DMSO, aglycerol, a glycol, a propylene glycol, an ethylene glycol, or acombination thereof. Optionally, the cryopreservation medium comprisesone or more non cell-permeating cryopreservatives selected frompolyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, amonosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose,a dextran, or a combination thereof. Other examples of usefulcryopreservatives are described in “Cryopreservation” (BioFiles Volume 5Number 4—Sigma-Aldrich® datasheet).

In one embodiment, the cryopreservation medium comprises acell-permeating cryopreservative, wherein the majority of thecell-permeating cryopreservative is DMSO. Optionally, thecryopreservation medium does not comprise a substantial amount ofglycerol.

In one embodiment, the cryopreservation medium comprises DMSO.Optionally, the cryopreservation medium does not comprise glycerol in amajority amount. Optionally, the cryopreservation medium does notcomprise a substantial amount of glycerol.

In one embodiment, the cryopreservation medium comprises additionalcomponents such as albumin (e.g. HSA or BSA), an electrolyte solution(e.g. Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 15%albumin by weight and about 5% to about 20% cryopreservative by volume(e.g. about 10%). Optionally, the cryopreservative comprises DMSO (e.g.in a majority amount).

In one embodiment, cryopreservation comprises placing the placenta onnitrocellulose paper.

In one embodiment, the placenta is cut into a plurality of sectionsbefore cryopreservation. Optionally, the sections are placed onnitrocellulose paper before refrigeration.

Methods of Use

The placental products of the present invention may be used to treat anytissue injury. A method of treatment may be provided, for example, byadministering to a subject in need thereof, a placental product of thepresent invention.

A typical administration method of the present invention is topicaladministration. Administering the present invention can optionallyinvolve administration to an internal tissue where access is gained by asurgical procedure.

Placental products can be administered autologously, allogeneically orxenogeneically.

In one embodiment, a present placental product is administered to asubject to treat a wound. Optionally, the wound is a laceration, scrape,thermal or chemical burn, incision, puncture, or wound caused by aprojectile. Optionally, the wound is an epidermal wound, skin wound,chronic wound, acute wound, external wound, internal wounds, congenitalwound, ulcer, or pressure ulcer. Such wounds may be accidental ordeliberate, e.g., wounds caused during or as an adjunct to a surgicalprocedure. Optionally, the wound is closed surgically prior toadministration.

In one embodiment, a present placental product is administered to asubject to treat a burn. Optionally, the burn is a first-degree burn,second-degree burn (partial thickness burns), third degree burn (fullthickness burns), infection of burn wound, infection of excised andunexcised burn wound, loss of epithelium from a previously grafted orhealed burn, or burn wound impetigo.

In one embodiment, a present placental product is administered to asubject to treat an ulcer, for example, a diabetic ulcer (e.g. footulcer).

In one embodiment, a placental product is administered by placing theplacental product directly over the skin of the subject, e.g., on thestratum corneum, on the site of the wound, so that the wound is covered,for example, using an adhesive tape. Additionally or alternatively, theplacental product may be administered as an implant, e.g., as asubcutaneous implant.

In one embodiment, a placental product is administered to the epidermisto reduce rhtids or other features of aging skin. Such treatment is alsousefully combined with so-called cosmetic surgery (e.g. rhinoplasty,rhytidectomy, etc.).

In one embodiment, a placental product is administered to the epidermisto accelerate healing associated with a dermal ablation procedure or adermal abrasion procedure (e.g. including laser ablation, thermalablation, electric ablation, deep dermal ablation, sub-dermal ablation,fractional ablation, and microdermal abrasion).

Other pathologies that may be treated with placental products of thepresent invention include traumatic wounds (e.g. civilian and militarywounds), surgical scars and wounds, spinal fusions, spinal cord injury,avascular necrosis, reconstructive surgeries, ablations, and ischemia.

In one embodiment, a placental product of the present invention is usedin a tissue graft procedure. Optionally, the placental product isapplied to a portion of the graft which is then attached to a biologicalsubstrate (e.g. to promote healing and/or attachment to the substrate).By way of non-limiting example, tissues such as skin, cartilage,ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenterand sinew can be used as tissue graft.

In one embodiment, a placental product is used in a tendon or ligamentsurgery to promote healing of a tendon or ligament. Optionally, theplacental product is applied to portion of a tendon or ligament which isattached to a bone. The surgery can be any tendon or ligament surgery,including, e.g. knee surgery, shoulder, leg surgery, arm surgery, elbowsurgery, finger surgery, hand surgery, wrist surgery, toe surgery, footsurgery, ankle surgery, and the like. For example, the placental productcan be applied to a tendon or ligament in a grafting or reconstructionprocedure to promote fixation of the tendon or ligament to a bone.

Through the insight of the inventors, it has surprisingly beendiscovered that placental products of the present invention providesuperior treatment (e.g. healing time and/or healing strength) fortendon and ligament surgeries. Tendon and ligament surgeries can involvethe fixation of the tendon or ligament to bone. Without being bound bytheory, the present inventors believe that osteogenic and/orchondrogenic potential of MSCs in the present placental productspromotes healing process and healing strength of tendons or ligaments.The present inventors believe that the present placental productsprovide an alternative or adjunctive treatment to periosteum-basedtherapies. For example, useful periosteum based treatments are describedin Chen et al. (“Enveloping the tendon graft with periosteum to enhancetendon-bone healing in a bone tunnel: A biomechanical and histologicstudy in rabbits”; Arthroscopy. March; 19(3):290-6), Chen et al.(“Enveloping of periosteum on the hamstring tendon graft in anteriorcruciate ligament reconstruction”; Arthroscopy. 2002 May-June;18(5):27E), Chang et al. (“Rotator cuff repair with periosteum forenhancing tendon-bone healing: a biomechanical and histological study inrabbits”; Knee Surgery, Sports Traumatology, Arthroscopy Volume 17,Number 12, 1447-1453), each of which are incorporated by reference.

As non-limiting example of a method of tendon or ligament surgery, atendon is sutured to and/or wrapped or enveloped in a placental membraneand the tendon is attached to a bone. Optionally, the tendon is placedinto a bone tunnel before attached to the bone.

In one embodiment, the tendon or ligament surgery is a graft procedure,wherein the placental product is applied to the graft. Optionally, thegraft is an allograft, xenograft, or an autologous graft.

In one embodiment, the tendon or ligament surgery is repair of a tornligament or tendon, wherein the placental product is applied to the tornligament or tendon.

Non-limiting examples of tendons to which a placental product can beapplied include a digitorum extensor tendon, a hamstring tendon, a biceptendon, an Achilles Tendon, an extensor tendon, and a rotator cufftendon.

In one embodiment, a placental product of the present invention is usedto reduce fibrosis by applying the placental product to a wound site.

In one embodiment, a placental product of the present invention is usedas an anti-adhesion wound barrier, wherein the placental product isapplied to a wound site, for example, to reduce fibrosis (e.g.postoperative fibrosis).

Non-limiting examples of wound sites to which the placental product canbe applied include those that are surgically induced or associated withsurgery involving the spine, laminectomy, knee, shoulder, or childbirth, trauma related wounds or injuries, cardiovascular procedures,angiogenesis stimulation, brain/neurological procedures, burn and woundcare, and ophthalmic procedures. For example, optionally, the wound siteis associated with surgery of the spine and the stromal side of theplacental product is applied to the dura (e.g. the stromal side facingthe dura). Direction for such procedures, including the selection ofwound sites and/or methodologies, can be found, for example, in WO2009/132186 and US 2010/0098743, which are hereby incorporated byreference.

A placental product of the present invention can optionally be used toreduce adhesion or fibrosis of a wound. Postoperative fibrosis is anatural consequence of all surgical wound healing. By example,postoperative peridural adhesion results in tethering, traction, andcompression of the thecal sac and nerve roots, which cause a recurrenceof hyperesthesia that typically manifests a few months after laminectomysurgery. Repeated surgery for removal of scar tissue is associated withpoor outcome and increased risk of injury because of the difficulty ofidentifying neural structures that are surrounded by scar tissue.Therefore, experimental and clinical studies have primarily focused onpreventing the adhesion of scar tissue to the dura matter and nerveroots. Spinal adhesions have been implicated as a major contributingfactor in failure of spine surgery. Fibrotic scar tissue can causecompression and tethering of nerve roots, which can be associated withrecurrent pain and physical impairment.

Without being bound by theory, the present inventors believe thatplacental products taught herein are useful to reduce adhesion orfibrosis of a wound, at least in part, because the placental productscan perform the very critical function in-situ of providing aimmunoprivileged environment (i.e. relatively high resistance againstimmune responses) in the human development process. One advantage of thewound dressings and processes of the present invention is that ananti-adhesion barrier is provided which can be used to prevent adhesionsfollowing surgery, and in particular following back surgery.

In the preceding paragraphs, use of the singular may include the pluralexcept where specifically indicated. As used herein, the words “a,”“an,” and “the” mean “one or more,” unless otherwise specified. Inaddition, where aspects of the present technology are described withreference to lists of alternatives, the technology includes anyindividual member or subgroup of the list of alternatives and anycombinations of one or more thereof.

The disclosures of all patents and publications, including publishedpatent applications, are hereby incorporated by reference in theirentireties to the same extent as if each patent and publication werespecifically and individually incorporated by reference.

It is to be understood that the scope of the present technology is notto be limited to the specific embodiments described above. The presenttechnology may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

Likewise, the following examples are presented in order to more fullyillustrate the present technology. They should in no way be construed,however, as limiting the broad scope of the technology disclosed herein.

The presently described technology and its advantages will be betterunderstood by reference to the following examples. These examples areprovided to describe specific embodiments of the present technology. Byproviding these specific examples, it is not intended limit the scopeand spirit of the present technology. It will be understood by thoseskilled in the art that the full scope of the presently describedtechnology encompasses the subject matter defined by the claimsappending this specification, and any alterations, modifications, orequivalents of those claims.

EXAMPLES

Other features and embodiments of the present technology will becomeapparent from the following examples which are given for illustration ofthe present technology rather than for limiting its intended scope.

Example 1 Characterization of Placental Membranes

Cells in placental membranes were characterized by FluorescenceActivated Cell Sorting (FACS) demonstrated the presence of stromal cells(Mesenchymal Stem Cell-like cells) in addition to fetal epithelial cellsand fibroblasts.

One unique characteristic of the presently disclosed placental productsis the presence of MSCs, which have been shown to be one of three typesof cells (in addition to epithelial cells and fibroblasts) that areimportant for wound healing. Placental membranes secrete a variety offactors involved in wound healing such as angiogenic factors, factorssupporting proliferation and migration of epithelial cells andfibroblasts, factors attracting endothelial stem cells from bloodcirculation to the wound site, antibacterial factors, and others.

Evaluation of proteins secreted by exemplary placental products of theinvention in comparison to Apligraf and Dermagraft demonstrated a numberof growth factors present in the tested products that are involved inwound healing. Examples are Vascular Endothelial Growth Factor (VEGF),Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor (TGF)and others. However, several unique factors including Epidermal GrowthFactor (EGF), which is one of the key factors for wound healing, arepresent in placental membranes and absent in Apligraf and Dermagraft.Also, placental membranes have a favorable protease-to-proteaseinhibitor ratio for wound healing. In an in vitro model of wound healing(cell migration assay, disclosed herein), the present inventors havedemonstrated that placental membranes secrete factors promoting cellmigration that will support wound closure.

Example 2 Exemplary Manufacturing Process of a Placental Product

In one embodiment, the present invention is a method of manufacturing aplacental product comprising an amniotic membrane and optionally achorionic membrane from placenta post partum. One such method is:

-   a. Remove umbilical cord close to placental surface,-   b. Blunt dissect of the amnion to placental skirt,-   c. Flip placenta over and completely remove amnion,-   d. Rinse amnion in PBS to remove red blood cells,-   e. Rinse amnion once with 11% ACD-A solution to assist in red blood    cell removal,-   f. Rinse amnion with PBS to remove ACD-A solution,-   g. Use PBS to remove any remaining blood from the amnion,-   h. Gently remove the connective tissue layer from the amnion,-   i. Place the amnion in PBS and set aside,-   j. Place the amnion into a bottle containing antibiotic solution and    incubate at 37° C. ±2° C. for 24-28 hrs,-   k. Remove bottle from the incubator and rinse membrane with PBS to    remove antibiotic solution,-   l. Mount amnion (epithelial side up) on reinforced nitrocellulose    paper and cut to size,-   m. Place into an FP-90 cryobag and heat seal,-   n. Add 50 mL cryopreservation solution to the bag through a syringe    and remove any air trapped within the bag with the syringe,-   o. Tube seal the solution line on the FP-90 bag,-   P. Place filled bag into secondary bag and heat seal,-   q. Place unit into packaging carton,-   r. Refrigerate at 2-8° C. for 30-60 minutes, Freeze at −80° C.±5° C.    inside a Styrofoam container.

Example 3 Exemplary Manufacturing Process of a Placental ProductContaining an Amniotic Membrane and a Chorionic Membrane

In one embodiment, the present invention is a method of manufacturing aplacental product comprising an amniotic membrane and optionally achorionic membranes from placenta post partum. One such method is:

-   a. Remove umbilical cord close to placental surface,-   b. Blunt dissect of the amnion to placental skirt,-   c. Flip placenta over and completely remove amnion,-   d. Remove chorion by cutting around placental skirt,-   e. Rinse both membranes in PBS to remove red blood cells,-   f. Rinse both membranes once with 11% ACD-A solution to assist in    red blood cell removal,-   g. Rinse both membranes with PBS to remove ACD-A solution, \-   h. Treat chorion in 0.5% dispase solution at 37° C.±2° C. for 30-45    minutes, optionally, during dispase incubation period, use PBS to    remove any remaining blood from the amnion,-   i. Gently remove the connective tissue layer from the amnion,-   j. Place the amnion in PBS and set aside,-   k. When dispase treatment is complete, rinse chorion with PBS to    remove dispase solution,-   l. Gently remove trophoblast layer from the chorion,-   m. Place the amnion and chorion each into a bottle containing    antibiotic solution and incubate at 37° C.±2° C. for 24-28 hrs,-   n. Remove bottles from the incubator and rinse each membrane with    PBS to remove antibiotic solution,-   o. Mount amnion (epithelial side up) or chorion on reinforced    nitrocellulose paper and cut to size,-   P. Place each piece into an FP-90 cryobag and heat seal,-   q. Add 50 mL cryopreservation solution to the bag through a syringe    and remove any air trapped within the bag with the syringe,-   r. Tube seal the solution line on the FP-90 bag,-   s. Place filled bag into secondary bag and heat seal,-   t. Place unit into packaging carton,-   u. Refrigerate at 2-8° C. for 30-60 minutes,-   v. Freeze at −80° C.±5° C. inside a Styrofoam container.

Example 4 Exemplary Placental Product Manufacturing Process

One method manufacturing a placental product comprising an amnioticmembrane according to the presently disclosed manufacturing procedure isas follows:

One method of manufacturing a placental product comprising a chorionicmembrane product and an amniotic membrane product according to thepresently disclosed manufacturing procedure was as follows:

The placenta was processed inside a biological safety cabinet. Theumbilical cord was first removed, and the amniotic membrane was peeledfrom the underlying chorionic membrane using blunt dissection. Themembrane was rinsed with phosphate buffered saline (PBS) (GibcoInvitrogen, Grand Island, N.Y.) to remove gross blood clots and anyexcess blood cells. The membrane was then washed with 11% anticoagulantcitrate dextrose solution (USP) formula A (ACD-A) (Baxter HealthcareCorp., Deerfield, Ill.) in saline (Baxter Healthcare Corp., Deerfield,Ill.) to remove remaining blood cells.

The stromal side of the amnion was cleaned by gently scraping away anyremaining connective tissue.

The amnion was then each disinfected in 500 mL of antibiotic solutionconsisting of gentamicin sulfate (50 μg/mL) (Abraxis PharmaceuticalProducts, Schaumburg, Ill.), vancomycin HCl (50 μg/mL) (Hospira Inc.,Lake Forest, Ill.), and amphotericin B (2.5 μg/mL) (Sigma Aldrich, St.Louis, Mo.) in DMEM at 37° C.±2° C. for 24-28 hours. Vented caps wereused with the incubation flasks. After the incubation period, themembrane was washed with PBS to remove any residual antibiotic solution.

The membrane was mounted on Optitran BA-S 85 reinforced nitrocellulosepaper (Whatman, Dassel, Germany) and cut to the appropriate size priorto packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem,N.C.). The stromal side of the amnion was mounted towards thenitrocellulose paper. Once the membrane unit was placed into the FP-90cryobag and the cryobag was heat sealed, 50 mL of a cryopreservationsolution containing 10% dimethyl sulfoxide (DMSO) (Bioniche Teo. InverinCo., Galway, Ireland) and 5% human serum albumin (HSA) (Baxter, WestLake Village, Calif.) in PlasmaLyte-A (Baxter Healthcare Corp.,Deerfield, Ill.) were added through the center tubing line. Any excessair was removed, and the tubing line was subsequently sealed.

The FP-90 cryobag was placed into a mangar bag (10 in.×6 in.) (MangarIndustries, New Britain, Pa.), which was then heat sealed. The mangarbag was placed into a packaging carton (10.5 in.×6.5 in.×0.6 in.)(Diamond Packaging, Rochester, N.Y.). All cartons were refrigerated at2-8° C. for 30-60 minutes prior to freezing at −80° C.±5° C. inside aStyrofoam container.

Example 5 Exemplary Manufacturing Process of a Placental ProductComprising Chorionic Membrane and Amniotic Membrane

One method of manufacturing a placental product comprising a chorionicmembrane product and an amniotic membrane product according to thepresently disclosed manufacturing procedure was as follows:

The placenta was processed inside a biological safety cabinet. Theumbilical cord was first removed, and the amniotic membrane was peeledfrom the underlying chorionic membrane using blunt dissection.Subsequently, the chorion was removed by cutting around the placentalskirt on the side opposite of the umbilical cord. The chorion on theumbilical side of the placenta was not removed due to thevascularization on this side. Both membranes were rinsed with phosphatebuffered saline (PBS) (Gibco Invitrogen, Grand Island, N.Y.) to removegross blood clots and any excess blood cells. The membranes were thenwashed with 11% anticoagulant citrate dextrose solution (USP) formula A(ACD-A) (Baxter Healthcare Corp., Deerfield, Ill.) in saline (BaxterHealthcare Corp., Deerfield, Ill.) to remove remaining blood cells.

The chorion was then incubated in 200 mL of a 0.5% dispase (BDBiosciences, Bedford, Mass.) solution in Dulbecco's modified eaglesmedia (DMEM) (Lonza, Walkersville, Md.) at 37° C.±2° C. for 30-45minutes to digest the connective tissue layer between the chorion andadjacent trophoblast layer. During this incubation period, the stromalside of the amnion was cleaned by gently scraping away any remainingconnective tissue. Once the chorion incubation period was complete, thechorion was rinsed with PBS to remove the dispase solution.Subsequently, the trophoblast layer was removed by gently peeling orscraping away these maternal decidual cells.

The amnion and chorion were then each disinfected in 500 mL ofantibiotic solution consisting of gentamicin sulfate (50 μg/mL) (AbraxisPharmaceutical Products, Schaumburg, Ill.), vancomycin HCl (50 μg/mL)(Hospira Inc., Lake Forest, Ill.), and amphotericin B (2.5 μg/mL) (SigmaAldrich, St. Louis, Mo.) in DMEM at 37° C.±2° C. for 24-28 hours. Ventedcaps were used with the incubation flasks. After the incubation period,the membranes were washed with PBS to remove any residual antibioticsolution.

The membranes were mounted on Optitran BA-S 85 reinforced nitrocellulosepaper (Whatman, Dassel, Germany) and cut to the appropriate size priorto packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem,N.C.). For the amnion, the stromal side was mounted towards thenitrocellulose paper. Once a membrane unit was placed into the FP-90cryobag and the cryobag was heat sealed, 50 mL of a cryopreservationsolution containing 10% dimethyl sulfoxide (DMSO) (Bioniche Teo. InverinCo., Galway, Ireland) and 5% human serum albumin (HSA) (Baxter, WestLake Village, Calif.) in PlasmaLyte-A (Baxter Healthcare Corp.,Deerfield, Ill.) were added through the center tubing line. Any excessair was removed, and the tubing line was subsequently sealed.

The FP-90 cryobag was placed into a mangar bag (10 in.×6 in.) (MangarIndustries, New Britain, Pa.), which was then heat sealed. The mangarbag was placed into a packaging carton (10.5 in.×6.5 in.×0.6 in.)(Diamond Packaging, Rochester, N.Y.). All cartons were refrigerated at2-8° C. for 30-60 minutes prior to freezing at −80° C.±5° C. inside aStyrofoam container.

Example 6 Quantitative Evaluation of Cell Number and Cell Viabilityafter Enzymatic Digestion of Placental Membranes

Amnion and chorion membranes and present placental products (from above)were evaluated for cell number and cell viability throughout theprocess. These analyses were performed on fresh placental tissue (priorto the antibiotic treatment step), placental tissue post antibiotictreatment, and product units post thaw. Cells were isolated from theplacental membranes using enzymatic digestion. For the frozen productunits, the FP-90 cryobags were first removed from the packaging cartonsand mangar bags. Then the FP-90 cryobags were thawed for 2-3 minutes ina room temperature water bath. Early experiments involved the use of a37° C.±2° C. water bath. After thaw, the placental membranes wereremoved from the FP-90 cryobag and placed into a reservoir containingsaline (Baxter Healthcare Corp., Deerfield, Ill.) for a minimum of 1minute and a maximum of 60 minutes. Each membrane was detached from thereinforced nitrocellulose paper prior to digestion.

Amniotic membranes were digested with 40 mL of 0.75% collagenase(Worthington Biochemical Corp., Lakewood, N.J.) solution at 37° C.±2° C.for 20-40 minutes on a rocker. After collagenase digestion, the sampleswere centrifuged at 2000 rpm for 10 minutes. The supernatant wasremoved, and 40 mL of 0.05% trypsin-EDTA (Lonza, Walkersville, Md.) wereadded and incubated at 37° C.±2° C. for an additional 5-15 minutes on arocker. The trypsin was warmed to 37° C.±2° C. in a water bath prior touse. After trypsin digestion, the suspension was filtered through a 100μm cell strainer nylon filter to remove any debris. Centrifugation at2000 rpm for 10 minutes was performed, and supernatant was removed. Cellpellets were reconstituted with a volume of PlasmaLyte-A that wasproportional to the pellet size, and 20 μL of the resuspended cellsuspension were mixed with 80 μL of trypan blue (Sigma Aldrich, St.Louis, Mo.) for counting. The cell count sample was placed into ahemocytometer and evaluated using a microscope.

Chorionic membranes were digested with 25 mL of 0.75% collagenasesolution at 37° C.±2° C. for 20-40 minutes on a rocker. Aftercollagenase digestion, the suspension was filtered through a 100 μm cellstrainer nylon filter to remove any debris. Centrifugation at 2000 rpmfor 10 minutes was performed, and supernatant was removed. Cell pelletswere reconstituted with a volume of PlasmaLyte-A that was proportionalto the pellet size, and 20 μL of the resuspended cell suspension weremixed with 80 μL of trypan blue for counting. The cell count sample wasplaced into a hemocytometer and evaluated using a microscope.

Placenta membranes were analyzed prior to any processing to determinethe initial characteristics of the membranes. Table 1 contains theaverage cell count per cm² and cell viability values for the amnioticand chorionic membranes from 32 placenta lots.

The average cell count per cm² for the amniotic membrane was 91,381cells with a corresponding average cell viability of 84.5%. For thechorionic membrane, the average cell count per cm² was 51,614 cells witha corresponding cell viability of 86.0%.

These data illustrate cell numbers that are useful with certainembodiments of the present invention; e.g. placental product comprisingan amniotic membrane containing about 10,000 to about 360,000 cells/cm².Since the amniotic membrane consists of epithelial cells and stromalcells, experiments were conducted to determine the ratio of epithelialcells to stromal cells. Amniotic membranes from 3 placenta lots wereanalyzed. First, a 5 cm×5 cm piece of amniotic membrane was digestedwith approximately 25 mL of 0.05% trypsin-EDTA (Lonza, Walkersville,Md.) at 37° C.±2° C. in a water bath for 30 minutes. After theincubation step, epithelial cells were removed by gently scraping thecells from the membrane. After rinsing with PBS (Gibco Invitrogen, GrandIsland, N.Y.), the membrane was subsequently digested in the same manneras chorionic membrane (described above). In addition, another intact 5cm×5 cm piece of amniotic membrane was digested using the standardprocedure (described above) to determine the total number of cells. Thepercentage of stromal cells was then determined by dividing the cellcount from the amniotic membrane with the epithelial cells removed withthe cell count from the intact membrane.

Results indicate that 19% of the total cells were stromal cells.Therefore, approximately 17,362 stromal cells were present in amnioticmembrane with approximately 74,019 epithelial cells. These dataindicated that there are approximately 3 times more stromal cells inchorionic membranes as compared to amniotic membranes. This ratio isconsistent with certain embodiments of the present invention thatprovide a placental product comprising a chorionic membrane and anamniotic membrane, wherein the chorionic membrane comprises about 2 toabout 4 times more stromal cells relative to the amniotic membrane.Table 1 Cell count per cm² and cell viability values from freshplacental tissue from 32 donors.

Membrane Statistics Cell Count per cm² Cell Viability Amnion Average91,381 84.5% SD 49,597 3.7% Chorion Average 51,614 86.0% SD 25,478 4.7%

Cell count and cell viability was assessed after the antibiotictreatment step. Table 2 provides the results from these analyses. Cellrecoveries from this step for the amniotic membrane and the chorionicmembrane were 87.7% and 70.3%, respectively.

TABLE 2 Cell count per cm², cell viability, and process (antibiotictreatm^(e)nt) cell recovery values for post antibiotic placental tissuefrom 28 donors. Cell Count per Process Cell Membrane Statistics cm² CellViability Recovery Amnion Average 75,230 84.4% 87.7% SD 46,890 4.2%49.4% Chorion Average 33,028 85.6% 70.3% SD 18,595 4.4% 31.1%

Example 7 Development of a Placental Product Cryopreservation Procedure

Cryopreservation is a method that provides a source of tissues andliving cells. A main objective of cryopreservation is to minimize damageto biological materials during low temperature freezing and storage.Although general cryopreservation rules are applicable to all cells,tissues, and organs, optimization of the cryopreservation procedure isrequired for each type of biological material. The present applicationdiscloses a cryopreservation procedure for placental membrane productsthat can selectively deplete immunogenic cells from the placentalmembranes; and preserve viability of other beneficial cells that are theprimary source of factors for the promotion of healing.

During cryopreservation method development for placental membranes, thepresent inventors evaluated key parameters of cryopreservation includingvolume of cryopreservative solution, effect of tissue equilibrationprior to freezing, and cooling rates for a freezing procedures.

Acceptance of tissue allografts in the absence of immunosuppression willdepend on the number of satellite immune cells present in the tissue.Cryopreservation is an approach which can be utilized to reduce tissueimmunogenicity. This approach is based on differential susceptibility ofdifferent cell types to freezing injury in the presence of DMSO;leukocytes are sensitive to fast cooling rates. The freezing rate of 1°C./min is considered optimal for cells and tissues including immunecells. Rapid freezing rates such as 60-100° C./min eliminate immunecells. However, this type of procedure is harmful to other tissue cells,which are desirable for preservation according to the present invention.The developed cryopreservation procedure utilized a cryopreservationmedium containing 10% DMSO, which is a key component protecting cellsfrom destruction when water forms crystals at low temperatures. Thesecond step of cryopreservation was full equilibration of placentalmembrane in the cryopreservation medium, which was achieved by soakingmembranes in the cryopreservation medium for 30-60 min at 4° C. Thisstep allowed DMSO to penetrate the placental tissues. Although there aredata in the literature showing that tissue equilibration prior tofreezing affects survival of immune cells (Taylor & Bank, Cryobiology,1988, 25:1),

It was unexpectedly found that 30-60 min placental membraneequilibration in a DMSO-containing solution at 2-8° C. increasessensitivity of immune cells to freezing so that these type of cellscannot withstand freezing even at a 1° C./min freezing rate.

Temperature mapping experiments were performed to analyze thetemperature profiles of potential cryopreservation conditions for themembrane products. These results are illustrated in FIG. 1. Eight (8)FP-90 cryobags were filled with either 20 mL or 50 mL ofcryopreservation solution, and temperature probes were placed insideeach cryobag. The first set of parameters (conditions 1 through 4 ofFIG. 1a through FIG. 1d , respectively) involved a 30-minuterefrigeration (2-8° C.) step prior to freezing (−80° C.±5° C.). Inaddition, the analysis involved freezing of the cryobags either inside aStyrofoam container or on the freezer shelf. The second set ofparameters (conditions 5 through 8 of FIG. 1e through FIG. 1h ,respectively) involved direct freezing (−80° C.±5° C.) of the cryobagseither inside a Styrofoam container or on the freezer shelf. The resultsindicated that condition 6 and condition 2 exhibited the most gradualtemperature decreases. Gradual temperature decreases are typicallydesired in order to preserve cell viability. The difference betweencondition 6 and condition 2 was that condition 2 included a 30-minuterefrigeration step. Therefore, the decrease in temperature from thestart of freezing to −4° C., where latent heat evolution upon freezingoccurs, was examined further. For condition 6, the rate of cooling wasapproximately −1° C./minute during this period. The rate of cooling forcondition 2 was approximately −0.4° C./minute during the same timeframe.Therefore, condition 2 was selected for incorporation into anon-limiting cryopreservation process since slower rates of cooling aregenerally desired to maintain optimal cell viability.

FIG. 2 depicts the effects of cryopreservation solution volume onprocess (cryopreservation) cell recovery for the amniotic membrane. Theanalysis of the 10 mL cryopreservation solution volume involved 5placenta lots, and the analysis of the 20 mL cryopreservation solutionvolume included 3 lots. For the 50 mL cryopreservation solution volume,14 placenta lots were analyzed.

As depicted in FIG. 2, the 50 mL volume of cryopreservation solutionvolume provided superior cell recovery compared to that of the 10 ml and20 ml. These data indicate that a cryopreservation medium volume ofgreater than 20 mL such as about 50 mL or more can provide superiorplacental product according to the present invention.

FIG. 3 shows the results of a similar study of analysis of thecryopreservation cell recovery for the chorionic membrane, demonstratingthat a cryopreservation solution volume of 50 mL was optimal. Theanalysis of the 10 mL cryopreservation solution volume involved 5placenta lots, and the analysis of the 20 mL cryopreservation solutionvolume included 3 lots. For the 50 mL cryopreservation solution volume,16 placenta lots were analyzed.

Experiments were conducted to evaluate different potential freezingconditions to maximize cell recovery after the cryopreservation process.FIG. 4 (cells from amniotic membrane) and FIG. 5 (cells from chorionicmembranes) depict these results, showing the effects of refrigerationtime and freezing parameters on process (cryopreservation) cell recoveryfor the chorionic membrane. Three conditions were analyzed. Theseconditions were also linked to the temperature mapping studies. Thefirst condition involved directly freezing the product unit on a shelfwithin the freezer (−80° C.±5° C.). The second condition also containeda direct freeze, but the product unit was placed into a Styrofoamcontainer within the freezer. The third condition included arefrigeration (2-8° C.) period of 30 minutes prior to the freezing step.For the amniotic membrane, 3 placenta lots were evaluated. Two (2)placenta lots were analyzed for the chorionic membrane. Resultsindicated that the third condition was optimal for both membrane types.

All of the cryopreservation parameters that were assessed for theamniotic and chorionic membranes are summarized in Table 3 and Table 4.The evaluation of the cell recoveries and cell viabilities from theseexperiments resulted in the selection of the final parameters for themanufacturing process. In addition, all average cell viability valueswere 70%.

TABLE 3 Post thaw cell count per cm², cell viability, and process(cryopreservation) cell recovery values for the amniotic membrane. CellProcess Condition Count Cell Cell Comments/ Parameter Tested Statisticsper cm² Viability Recovery Conclusions Refrigerate All Average 55,70983.4% 64.2% Overall at 2-8° C. for conditions SD 45,210 4.4% 22.5%assessment. 30-60 min N 32 32 32 and freeze at −80° C. ± 10° C.Refrigeration 30 min Average 52,173 83.1% 63.7% No significant timeinterval SD 39,750 4.5% 21.4% difference N 26 26 26 found in 60 minAverage 71,033 85.0% 66.5% process cell SD 66,525 3.9% 29.3% recovery. AN 6 6 6 30-60 min range was established. Thawing 37° C. ± Average 48,52483.3% 64.0% No significant temperature 2° C. water SD 27,804 1.7% 34.4%difference bath N 7 7 7 found in Room Average 57,721 83.5% 64.3% processcell temp SD 49,271 4.9% 19.0% recovery. The water bath N 25 25 25 roomtemp condition was selected for logistical reasons. Holding 1-15 minAverage 50,873 83.1% 65.0% No significant period after SD 38,969 3.9%24.2% difference transfer into N 26 26 26 found in saline 1 hr Average76,667 85.1% 61.0% process cell SD 66,565 6.2% 14.3% recovery. N 6 6 6Membranes can be held in saline for up to 1 hr. Tissue size 5 cm × 5Average 58,431 83.3% 62.8% No decrease cm SD 47,603 4.5% 21.7% inprocess cell N 28 28 28 recovery from 2 cm × 2 Average 36,656 84.4%73.9% the 5 cm × 5 cm SD 13,175 3.4% 29.5% cm product to N 4 4 4 the 2cm × 2 cm product. Both sizes were acceptable for use.

TABLE 4 Post thaw cell count per cm², cell viability, and process(cryopreservation) cell recovery values for the chorionic membrane. CellProcess Condition Count Cell Cell Parameter Tested Statistics per cm²Viability Recovery Refrigerate All Average 23,217 87.3% 102.8% at 2-8°C. for conditions SD 9,155 4.1% 65.5% 30-60 min N 27 27 27 and freeze at−80° C. ± 10° C. Dispase 30 min Average 22,354 85.7% 81.1% No decreasetreatment SD 9,505 5.1% 32.4% in process cell N 24 24 24 recovery for 45min Average 27,125 90.6% 172.6% the 45 min SD 7,963 2.2% 101.2%treatment. A N 6 6 6 30-45 min range was established. Refrigeration 30min Average 23,815 86.8% 102.2% The process time interval SD 9,681 5.2%68.8% recovery value N 25 25 25 was >80% for 60 min Average 20,773 85.8%84.9% the 60 min SD 7,356 4.7% 14.4% time interval. N 5 5 5 A 30-60 minrange was established. Thawing 37° C. ± Average 33,360 85.9% 114.7% Nosignificant temperature 2° C. water SD 8,497 4.0% 38.1% difference bathN 5 5 5 found in Room Average 21,298 86.8% 96.3% process cell temp SD8,189 5.3% 67.2% recovery. The water bath N 25 25 25 room temp conditionwas selected for logistical reasons. Holding 1-15 min Average 23,73386.6% 100.6% No significant period after SD 9,674 5.1% 67.0% differencetransfer into N 26 26 26 found in saline 1 hr Average 20,550 87.0% 91.4%process cell SD 6,575 4.8% 32.0% recovery. N 4 4 4 Membranes can be heldin saline for up to 1 hr. Tissue size 5 cm × 5 Average 23,391 86.1%99.6% No decrease cm SD 8,865 5.0% 58.7% in process cell N 23 23 23recovery from 2 cm × 2 Average 23,036 88.4% 98.7% the 5 cm × 5 cm SD11,362 5.0% 81.3% cm product to N 7 7 7 the 2 cm × 2 cm product. Bothsizes were acceptable for use. Notes: cm = centimeter; min = minutes;temp = temperature; hr = hour, SD = standard deviation; N = number.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising an amnioticmembrane containing about 40,000 to about 90,000 or to about 260,000cells/cm².

Example 8 Qualitative Evaluation of Cell Viability by Tissue Staining

The amniotic and chorionic membranes were stained using a LIVE/DEAD®Viability/Cytotoxicity kit (Molecular Probes Inc., Eugene, Oreg.) toqualitatively assess cell viability. Staining was performed as per themanufacturer's protocol. Membrane segments of approximately 0.5 cm×0.5cm were used. Evaluation of stained membranes was performed using afluorescent microscope. An intense uniform green fluorescence indicatedthe presence of live cells, and a bright red fluorescence indicated thepresence of dead cells. Images of fresh amniotic and chorionic membranesas well as cryopreserved amniotic and chorionic membranes demonstratedthat the manufacturing process did not alter the phenotypiccharacteristics of the membranes post thaw.

FIG. 6 contains representative images of fresh amniotic and chorionicmembranes as well as cryopreserved amniotic and chorionic membranes.These images demonstrated that the manufacturing process did not alterthe phenotypic characteristics of the membranes and the proportion ofviable cell types (epithelial and stromal cells) in the membranes postthaw.

FIG. 6 Representative images of the live/dead staining of the epitheliallayer of fresh amniotic membrane (A); epithelial layer of cryopreservedamniotic membrane (B); stromal layer of fresh amniotic membrane (C);stromal layer of cryopreserved amniotic membrane (D); fresh chorionicmembrane (E); and cryopreserved chorionic membrane (F).

Example 9 Placental Tissue Immunogenicity Testing

One unique feature of the human amnion and chorion is the absence offetal blood vessels that prevent mobilization of leukocytes from fetalcirculation. On the fetal side, macrophages resident in thechorioamniotic mesodermal layer represent the only population of immunecells. Thus, fetal macrophages present in the chorion and amnion are themajor source of tissue immunogenicity. However, the number ofmacrophages in amnion is significantly lower (Magatti et al, Stem Cells,2008, 26: 182), and this explains the low immunogenicity of amnion andthe ability to use it across HLA barriers without matching between thedonor and recipient (Akle et al, Lancet, 1981, 8254:1003; Ucakhan etal., Cornea, 2002, 21:169). In contrast, the chorion is consideredimmunogenic. In a study where the amnion was used together with thechorion for plastic repair of conjunctival defects, the success rate waslow (De Roth Arch Ophthalmol, 1940, 23: 522). Without being bound bytheory, the present inventors believe that removal of CD14+ cells fromplacental membranes eliminates activation of lymphocytes in vitro. Inaddition to the presence of fetal macrophages, the present inventorsbelieve that immunogenicity of chorion can be mediated by contaminationof blood cells coming from the maternal trophoblast, which containsblood vessels. Thus, the processing of placental membrane for clinicaluse can be enhanced by purification of the amnion and chorion frommaternal trophoblasts and selective elimination of all CD14+ fetalmacrophages.

Immunogenicity testing can be used to characterize a placental productas safe clinical therapeutics. For example, two bioassays can be used totest immunogenicity of manufactured placental products: Mixed LymphocyteReaction (MLR) and Lipopolysaccharide (LPS)-induced Tumor NecrosisFactor (TNF)-α secretion.

Example 10 Mixed Lymphocyte Reaction (MLR)

An MLR is a widely used in vitro assay to test cell and tissueimmunogenicity. The assay is based on the ability of immune cells(responders) derived from one individual to recognize allogeneic HumanLeukocyte Antigen (HLA) and other antigenic molecules expressed on thesurface of allogeneic cells and tissues (stimulators) derived fromanother individual when mixed together in a well of an experimentaltissue culture plate. The response of immune cells to stimulation byallogeneic cells and tissues can be measured using a variety of methodssuch as secretion of particular cytokines (e.g., Interleukin (IL)-2),expression of certain receptors (e.g., IL-2R), or cell proliferation,all of which are characteristics of activated immune cells.

Placental tissue samples representing different steps of the presentlydisclosed manufacturing process were used for immunogenicity testing.These samples included amnion with chorion and trophoblast as a startingmaterial and separated choriotrophoblast, chorion, trophoblast, andamnion. Both freshly purified and cryopreserved (final products) tissueswere tested.

For the MLR assay, cells from placental tissues were isolated using 280U/mL of collagenase type II (Worthington, Cat No. 4202). Tissues weretreated with enzyme for 60-90 min at 37° C.±2° C., and the resultingcell suspension was filtered through a 100 μm filter to remove tissuedebris. Single cell suspensions were then centrifuged using a Beckman,TJ-6 at 2000 rpm for 10 min and washed twice with DPBS. Supernatant wasdiscarded after each wash, and cells were resuspended in 2 mL of DMEM(Invitrogen, Cat No. 11885) and evaluated for cell number and cellviability by counting cells in the presence of Trypan blue dye(Invitrogen, Cat No. 15250-061). For the MLR, placental-derived cellswere mixed with allogeneic hPBMCs at a 1:5 ratio in 24-well cultureplates in DMEM supplemented with 5% fetal bovine serum (FBS) andincubated for 4 days in the incubator containing 5% CO₂, 95% humidity at37° C.±2° C. Human Peripheral Blood Mononuclear Cells (hPBMCs) alonewere used as a negative control, and a mixture of two sets of hPBMCsderived from two different donors was used as a positive MLR control.After 4 days of incubation, cells were collected from wells, lysed usinga lysis buffer (Sigma, Cat No. C2978) supplemented with proteaseinhibitor cocktail (Roche, Cat No. 11836153001), and IL-2R was measuredin cell lysates using the sIL-2R ELISA kit (R&D Systems, Cat No. SR2A00)according to the established protocol (G-SOP-Q088).

The level of IL-2R is a measure of activation of T-cells in response toimmunogenic molecules expressed by allogeneic cells. Results of 2 out of12 representative experiments are shown in FIG. 7. Results presented inthese figures demonstrate a method of manufacture of placentalmembranes, resulting in low immunogenicity of the final products.

As depicted in FIG. 7: the manufacturing process serially reducesimmunogenicity of the placental product. Samples representing differentsteps of the manufacturing process (Chorion+Trophoblast (CT),Trophoblast (T), Amnion (AM), Chorion (CM), and Amnion +Chorion (A/C))were co-cultured with hPBMCs for 4 days. IL-2aR was measured in celllysates as a marker of T-cell activation. Negative control shows a basallevel of immune cell activation: PBMCs derived from one donor werecultured alone. Positive control: a mixture of PBMCs derived from 2different donors.

As depicted in FIG. 8 selective depletion of immunogenicity results fromthe Fa3present cryopreservation process of producing the presentplacental products, as evidenced by the significant decrease inimmunogenicity upon cryopreservation.

Example 11 LPS-Induced TNF-α Secretion by Placental Membrane Cells

As described herein, fetal macrophages present in the amnion and chorionare a major source of tissue immunogenicity. Without being bound bytheory, the present inventors believe that removal of CD14+ cells fromplacental membrane eliminates activation of lymphocytes and thatdepletion of allogeneic donor tissue macrophages decreases the level ofinflammatory cytokine secretion and tissue immunogenicity. The inventorsalso believe that reduction of tissue immunogenicity can also be reachedby depletion of TNF-α with anti-TNF-α antibodies or suppression of TNF-αsecretion by IL-10. Macrophages in fetal placental membranes respond tobacteria by secretion of inflammatory cytokines. The secretion of TNF-αby fresh placental membranes in vitro in response to bacterial LPS issignificantly higher in the chorionic membrane. Thus, the presentinventors believe that immunogenicity of placental membranes is mediatedby macrophages, the amount and/or activity of which is higher in thechorionic membrane.

According to the present invention, selective depletion of macrophagesis an approach to selectively deplete immunogenicity of the amniotic andchorionic membranes, allowing the use of both allogeneic membranes forclinical applications. The assay of functional macrophages in aplacental product is used here as an assay for immunogenicity testing(e.g. in production or prior to clinical use) based on the facts that:macrophages are the source of immunogenicity in chorionic membranes.Macrophages in placenta-derived membranes respond to bacterial LPS bysecretion of high levels of TNF-α; and TNF-α is a critical cytokineinvolved in immune response and allograft tissue rejection. Therefore,secretion of TNF-α by placenta-derived membranes in response to LPS isused here to characterize tissue immunogenicity and for pre-usescreening.

Example 12 Establishment of Allowed LPS-Induced TNF-α Secretion Level byPlacental Membranes

Data from published reports regarding the level of TNF-α, which isassociated with the absence or an insignificant immune response in avariety of experimental systems, are presented in Table 5. These dataindicate that a TNF-α level below 100 pg/mL correlates with a low immuneresponse. The ability of amniotic and chorionic membranes to produceTNF-α spontaneously and in response to bacteria or bacterial LPS invitro has been shown by a number of investigators. Table 6 summarizessuch data. The lowest spontaneous TNF-α secretion by amniotic membraneof about 70 pg/cm² of the membrane was reported by Fortunato et al. Allreports also showed that fresh placental membranes secrete large amountsof TNF-α in response to bacteria or bacterial LPS, which is attributedto the presence of viable functional macrophages.

TABLE 5 TNF-α levels associated with no or an insignificant immuneresponse. TNF-α levels associated with the Description ofabsence/reduction experimental system of immune response CommentsReferences IL-10-induced inhibition Mean 260 pg/mL Wang et al., of MLRin vitro. Transplantation, TNF was measured in 2002, 74: 772 tissueculture supernatant by ELISA. MLR using skin tissue Mean 100 pg/mLexplants (0.02 cm2 per well) as stimulators in the presence or absenceof IL-10 (skin explant assay). Skin tissue destruction was assessedmicroscopically, and severity was assigned based on histopathologicaltissue damage. Endogeneous TNF ~0.04 U/mL for the TNF activity Shalabyet al, J production in MLR in the negative control and per mg is notImmunol, 1988, presence or absence of MLR in the presence provided. 141:499 anti-TNF antibodies. TNF of anti-TNF levels were assessedantibodies, which using the WEHI-164 correlated with no or cytotoxicityassay. significant inhibition of lymphocyte proliferation TNF levels inBAL fluid of Isograft: below Unmodified Sekine et al, J lung isografts,unmodified detection; allograft: ~45 pg/mL Immunol, 1997, allograft, andalveolar AM-depleted allograft: (immunogenic) 159: 4084. macrophages(AM) ~15 pg/mL of BAL depleted allograft in rats. (total 75 pg/5 ml ofBAL) TNF levels in MLR after ~<200 pg/mL TNF Ohashi et al, 48 hours inthe presence correlated with a Clin Immunol, or absence of advancedcomplete inhibition of 2009, e-pub glycation end products MLR ahead ofprint (MLR inhibitors). TNF levels in MLR. <100 pg/mL TNF in Toungouz etal, MLR with HLA- Hum Immunol, matched donors 1993, 38: 221 (control, nostimulation) TNF activity in MLR when Negative control ~20 Unit ofactivity Lomas et al, pieces of cryopreserved U of TNF activity; wascalculated Cell Tissue skin allografts (~0.2 cm²) MLR with skin as TNFin Bank, 2004, 5: were incubated with explants - 0-40 U; ng/mL divided23. hPBMCs for 24 hours. Positive control - 600 U by OD at 570 nmPositive control: for the hPBMC + LPS; negative - same hPBMC alone.experimental well Cytokine time course in Optimal TNF Jordan & Ritter,MLR, including TNF. after 24 hours - J Immunol ~150 pg/mL Meth, 2002,260: 1 MLR using skin tissue For no skin Recalculation Dickinson et al,explants (0.02 cm² per destruction: 0.5-1.1 pg/mL per 1 cm² of Cytokine,1994, well) as stimulators in the for HLA skin tissue: 6: 141 presenceor absence of compatible lowest TNF anti-TNF antibodies (skinresponders, and 2.6-1376 pg/mL non- explant assay). Skin for immunogenictissue destruction was unmatched MLR level is 100 pg/cm² assessedmicroscopically, and severity was assigned based on histopathologicaltissue damage.

TABLE 6 Secretion of TNF in vitro by fresh amniotic and chorionicmembranes. TNF levels Comments/ secreted by fresh recalculationsplacental of the lowest Description of membranes in TNF levels perexperimental system culture cm² References TNF secretion by Chorion:basal Lowest TNF Zaga et al., Biol “fresh” amnion and 3.3 ± 0.46 ng/cm²,level for amnion Reprod, 2004, chorion tissues (1.44 cm2) LPS-induced -150-250 ng/cm² is 1200 pg/cm² 71: 1296 incubated for 24 Amnion: basalhours in the presence 2.5 ± 1.3 ng/cm², or absence of LPS LPS-induced -~50 ng/cm² (500 ng/mL). TNF secretion by Basal ~1-2.5 pg/μg Lowest TNFZaga-Clavellina “fresh” amnion and total protein in the level for amnionet al, Reprod chorion tissues (1.8 cm medium for both is 800 pg/cm² BiolEndocrinol, diameter disks: 2.5 cm²) amnion and 2007, 5: 46 incubatedfor 24 chorion; hours in the presence E. Coli-induced: or absence of E.Coli amnion - 29.2 in 1 mL medium. (14.5-35.3) pg and chorion - 53.15(40-94.2) pg per μg total protein TNF secretion by Basal: ~2-64 U/mL 1unit = ~100-200 pg/mL; Paradowska et “fresh” amnion and or 8-10 mgchorion; Lowest TNF al, Placenta, chorion tissues <1 U/mL for 5-7 mglevel for amnion 1997, 18: 441 (chorion 8-10 mg amnion. is <100 pg/mLtissue/mL; amnion 5-7 mg/mL, LPS-induced: >100 corresponding to0.02-0.04 cm²) U/10 mg for chorion <2500 pg/cm² incubated for 20 hoursand ~15-17 U/10 mg in the presence or for amnion absence of LPS (5μg/mL). TNF secretion by Amnion: Basal - 40 pg/mL, Lowest TNF Fortunatoet al, “fresh” amnion (0.57 cm²) LPS-induced - 410 pg/mL level for freshAm J Obstet in 0.8 mL amnion is ~70 pg/cm² Gynecol, 1996, incubated for24 hours 174: 1855 in the presence or absence of LPS (50 ng/mL). TNFsecretion by Basal: Amnion ~7-13 ng/mL/g Amnion is 5-7 mg Thiex et al,“fresh” amnion and tissue); corresponds Reprod Biol chorion tissues (4cm²) Chorion ~18 ng/mL/g ~0.02-0.04 cm²; Endocrinol, incubated for 24tissue 1 g is ~6 cm²; 2009, 7: 117 hours in the presence LPS-induced(1000 ng/mL): Lowest TNF or absence of LPS (1-1000 ng/mL) Amnion levelfor amnion ~14 ng/mL/g), is ~1000 pg/cm² Chorion ~27 ng/mL/g

Example 13 LPS-Induced TNF-α Secretion and CT Induced MLR ImmunogenicityAssay

2 cm×2 cm pieces of placental membranes representing intermediates andfinal products were placed in tissue culture medium and exposed tobacterial LPS (1 μg/mL) for 20-24 hr. After 24 hours, tissue culturesupernatant were collected and tested for the presence of TNF-α using aTNF-α ELISA kit (R&D Systems) according to the manufacturer's protocol.Human hPBMCs (SeraCare) known to contain monocytes responding to LPS bysecretion of high levels of TNF-α were used as a positive control in theassay. hPBMCs and placental tissues without LPS were also included ascontrols in the analysis. In this assay, TNF detected in the culturemedium from greater than 70 pg/cm² (corresponding to 280 pg/mL) for bothspontaneous and LPS-induced TNF-α secretion was considered immunogenic.

As depicted in FIG. 9A and FIG. 9B, the manufacturing process seriallyreduces immunogenicity of the placental product. Samples representingdifferent steps of the manufacturing process (Amnion+Chorion+Trophoblast(ACT), Chorion+Trophoblast (CT), Amnion (AM), and Chorion (CM)) wereincubated in the presence of LPS for 24 hr, and after that tissueculture supernatants were tested for the TNF-α by ELISA. Tissuescultured in medium without LPS show the basal level of TNF a secretion.PBMCs, which are known to secrete high levels of TNF, were used as apositive control.

The low levels of TNF-α and the absence of the response to LPS by AM andCM indicates the absence of viable functional macrophages that are themajor source of immunogenicity for amniotic and chorionic membranes.Results of this assay showed a correlation with the MLR data: tissuesthat produce high levels of TNF-α in response to LPS are immunogenic inthe MLR assay (FIGS. 9A and 9B, for TNF-α secretion; FIG. 9, C-MLR).

As depicted in FIG. 9A and FIG. 9B, the manufacturing process seriallyreduces immunogenicity of the placental product. Samples representingdifferent steps of the manufacturing process (Amnion+Chorion+Trophoblast(ACT), Chorion+Trophoblast (CT), Amnion (AM), and Chorion (CM)) wereincubated in the presence of LPS for 24 hr, and after that tissueculture supernatants were tested for the TNF-α by ELISA. Tissuescultured in medium without LPS show the basal level of TNF a secretion.PBMCs, which are known to secrete high levels of TNF, were used as apositive control.

Choriotrophoblast (CT), which secreted high levels of TNF-α (FIG. 98),was tested in MLR against two different PBMC donors. CT cells wereco-cultured with PBMCs 0210 and 11706) for 4 days. IL-2αR was measuredin cell lysates as a marker of T-cell activation. Positive control: amixture of PBMCs derived from 2 different donors (1210 and 11706).

FIG. 9C shows that preparations producing high levels of TN F-α areimmunogenic. Choriotrophoblast (CT), which secreted high levels of TNF-α (FIG. 9B), was tested in MLR against two different PBMC donors. CTcells were co-cultured with PBMCs (1210 and 11706) for 4 days. IL-2αRwas measured in cell lysates as a marker of

T-cell activation. Positive control: a mixture of PBMCs derived from 2different donors (1210 and 11706).

Example 14 Analysis of Placental Cells by FACS

Knowing the cellular composition of amnion and chorionic membranes isimportant for developing a thorough understanding of potentialfunctional roles in wound healing and immunogenicity. Previous reportsdemonstrated that both amnion and chorion contains multiple cell types.Purified amnion has two major cellular layers: epithelial cells andstromal. In addition to epithelial cells and fibroblasts, stromal cellswere identified in the amnion and chorion. Although there are no fetalblood vessels within either the amniotic or chorionic membranes, bothmembranes comprise resident fetal macrophages. The close proximity tomaternal blood circulation and decidua provide a potential source ofimmunogenic cells (maternal leukocytes and trophoblast cells) andtherefore are a potential source of immunogenicity. To investigate thecellular composition of the amnion and chorion, FACS analysis wasperformed.

Example 14.1 FACS Procedure: Single Cell Suspension Preparation

Purified amnion and chorionic membranes were used for cellularphenotypic analysis via FACS. Cells from amnion and chorion wereisolated using 280 U/mL collagenase type II (Worthington, Cat No. 4202).Tissues were treated with enzyme for 60-90 min at 37° C.±2° C., and theresulting cell suspension was filtered through a 100 μm filter to removetissue debris. Single cell suspensions were then centrifuged using aBeckman TJ-6 at 2000 rpm for 10 min and washed twice with DPBS.Supernatant was discarded after each wash, and cells were resuspended in2 mL of FACS staining buffer (DPBS+0.09% NaN₃+1% FBS).

Example 14.2 Immunolabeling Cells for Specific Cellular Markers

Once the single cell suspension was prepared according to Example 10, aminimum of 1×10⁵ cells in 100 μL of FACS staining buffer was treatedwith antibodies labeled with fluorescent dye. Table 7 providesdescriptions of the antibodies and the amounts used. For cell surfacemarkers, cells were incubated for 30 min at room temperature in the darkwith antibodies followed by washing twice with FACS staining buffer bycentrifugation at 1300 rpm for 5 min using a Beckman TJ-6 centrifuge.Cells were then resuspended in 400 μL of FACS staining buffer andanalyzed using a BD FACSCalibur flow cytometer. To assess cellviability, 10 μL of 7-AAD regent (BD, Cat No. 559925) was added justafter the initial FACS analysis and analyzed again. For intracellularstaining, cells were permeabilized and labeled following themanufacturer's recommendations (BD Cytofix/Cytoperm, Cat No. 554714) andanalyzed using a BD FACSCalibur flow cytometer.

TABLE 7 Description of reagents used for placental cell characterizationby FACS. Volume of Cell marker antibody antibody and solution Cellmarker Cell marker label type Cat No. used type specificity IgG1 BD559320  5 μL Cell surface Isotype isotype-PE control CD105-PE Caltag 20μL Cell surface MSC marker MHCD10504 CD166-PE BD 559263 80 μL Cellsurface MSC marker CD45-PE BD 555483 10 μL Cell surface Hematopoeticcell marker IgG2a BD 555574  2 μL Cell surface Isotype controlisotype-PE CD14-PE BD 555398 20 μL Cell surface Monocyte markerHLA-DR-PE BD 556644 20 μL Cell surface HLA class II specific forantigen- presenting cells IgG1 BD555748  5 μL Cell surface Isotypecontrol isotype-FITC CD86-FITC BD 557343 20 μL Cell surface Immune co-stimulatory marker CD40-FITC BD 556624 20 μL Cell surface Immune co-stimulatory marker IgG1 Dako X0931 10 μL Intracellular Isotype controlisotype- unlabeled Cytokeratin Dako M7018  2 μL IntracellularTrophoblast 7-unlabeled marker Rabbit anti- Dako F0261  5 μLIntracellular Secondary mouse FITC antibody

Example 15 Phenotypic Analysis of Placental Cells

FACS analysis of single cell suspensions of both amnion and chorionmembranes demonstrates that both membranes contain cells expressingmarkers specific for mesenchymal stem cells (refer to Table 8),implicating the presence of MSCs. In addition, several immunogenicmarkers, which are more likely expressed on CD14+ placental macrophages,were detected. The % ranges for different markers are wide. It can beexplained by: 1) high variability in cell number between placentadonors; and 2) technical issues, which include the presence of high andvariable cellular and tissue debris in the cellular suspension. Althoughdebris can be gated out, debris particles that are comparable with cellsby size will affect the accuracy of the calculated % for each testedmarker. In addition, Table 9 provides a FACS analysis of cells isolatedfrom the amniotic and chorionic membranes that were cultured in 10% FBSin DMEM at 37° C.±2° C. until confluency (passage 0 cells).

These data demonstrated that cells derived from amniotic and chorionicmembranes retained a phenotype similar to MSCs after culturing. Inconclusion, the presence of stromal cells in placental tissues wasconfirmed by FACS analysis.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising an amnioticmembrane containing MSCs.

TABLE 8 Characterization of the cellular composition of placentalmembranes based on selective CD markers. Amnion (% Chorion (% Markerrange) range) MSC Markers CD105 72.1-88.2 6.4-78.5 CD166 17.3-58.04.8-51.5 Hematopoietic Cell CD14 6.93-10.5 0.9-6.1  Markers CD45 4.4-9.94.6-14.7 Immune co-stimulatory HLA-DR   0-5.6   0-14.7 markers CD8624.3-49.6 4.9-22.5 CD40  7.0-68.7  2-5.8 Trophoblast markerCytokeratin-7 1.36-4.66 2.71-23.07

TABLE 9 FACS analysis of cultured cells (passage 0) from placenta lotD16. Cell Surface Marker Amnion (%) Chorion (%) CD45 2.18 0.53 CD16692.77 82.62 CD105 83.02 86.73 CD49a 92.28 92.26 CD73 89.57 94.57 CD41a−0.03 −0.05 CD34 −0.23 −0.25 HLA-DR −0.23 −0.19 CD19 −0.19 −0.22 CD14−0.25 −0.27 CD90 99.12 98.00

Example 16 Aherence of Cells Derived from Placental Products

Therapeutic cells, in optional embodiment of the present invention, areadherent, express specific cellular markers such as CD105 and lackexpression of other markers such as CD45, and demonstrate the ability todifferentiate into adipocytes, osteoblasts, and

The expression of specific cellular markers has already been describedin Example 15. To determine if the cells within the placental productderived from the chorionic membrane can adhere to plastic anddifferentiate into one of the lineages, cells were isolated from theplacental product derived from the amnion as described in this inventionand cultured at 37° C.±2° C. and expanded.

FIG. 10 shows representative images of cells isolated and cultured fromamniotic (FIG. 10A) and chorionic (FIG. 10B) membranes demonstratingplastic adherence, which is a key feature of MSCs. As a comparison, arepresentative image of MSCs isolated and expanded from human bonemarrow aspirate is also provided (FIG. 10C). Together, these data showthat cells derived from amniotic and chorionic membranes retain aphenotype similar to MSCs after culturing as demonstrated by thecellular markers present in addition to the ability of the cells toadhere to plastic. In conclusion, the presence of MSCs in placentaltissues was confirmed by FACS analysis and tissue culture

Example 17 Live CD45+FACS Analysis

As CD45 is a general marker for hematopoietic cells and therefore amarker for the presence immunogenic cells, the presence of CD45+ cellsmay correlate well with how immunogenic a tissue may be. An initialstudy indeed showed a correlation between amount of immunogenicity asmeasured via an in vitro MLR assay of placental tissue at various stageswithin the manufacturing process (as described previously), and theamount of CD45+ cells was determined via FACS analysis. As FIG. 11demonstrates, membranes that trigger the expression of high levels ofIL-2sR on hPBMC responders in MLR also contained a high percentage ofCD45+ cells, indicating that immunogenicity of placental membranes canbe correlated with the number of CD45+ cells. Further studies revealed,however, that quantifying CD45+ cells via FACS alone showed highvariability that did not allow for the establishment of a safetythreshold for CD45+ cells in placental membranes. Accordingly, theinventors evaluated whether or not viability of CD45+ cells iscorrelated with immunogenicity.

To eliminate some of the variability in CD45+ measurements via FACS,viability of CD45+ cells was assessed, as dead CD45+ cells do notcontribute to immunogenicity. To ensure an accurate assessment of liveCD45+ cells, a pilot experiment was conducted in which a single cellsuspension of amnion membrane was spiked in with a known concentrationof live CD45+ cells (hPBMCs) ranging from a theoretical 1.25% to 20%(0.75-12%—actual % of the spiked cells) of the total cell concentrationin suspension. Cells were stained with CD45-PE antibody at determinedconcentrations (refer to Table 10), incubated with 7-AAD cell viabilitytest reagent, and analyzed using a BD FACSCalibur. Table 10 demonstratesthat recovery of known amounts of CD45+ cells was not correct (4thcolumn in the table). For example, although 12% of PBMCs was spiked intoa single-cell suspension of amnion membrane, only 4.26% of CD45+ cellswere recovered according to FACS analysis (>60% difference from theactual spike). To correlate with immunogenicity, MLR was also performedin parallel. Briefly, single cell suspensions of amniotic membranespiked with various amounts of live hPBMCs were co-cultured with anotherdonor of PBMCs in the MLR. FIG. 12 depicts a correlation between theamount of CD45+ cells present in amnion-derived cell suspensions andimmunogenicity in MLR in vitro. Table 10 and FIG. 12 show that thesuspensions spiked with higher amounts of live CD45+ cells resulted inhigher immunogenicity as measured by IL-2sR expression on the hPBMCresponder donor.

TABLE 10 % CD45+ recovery experiments. Cell Actual spike suspensionSample (%, based on % immunogenicity Description % CD45+ 60% CD45+Difference (tested in MLR (in % of cell cells cells in this from andexpressed types in the (detected hPBMC actual as IL-2R in mixture) byFACS) batch) spike pg/mL) 100% 0.65 N/A N/A 20.23 amnion 0% PBMC N/A N/AN/A 15.6 (negative control) 100% PBMC 61.51 N/A N/A 86.31 (positivecontrol) 20% 4.26 12 64.5% 24.38 PBMC + 80% Amnion 10% 2.24 6 62.7%21.17 PBMC + 90% Amnion 5% PBMC + 1.7 3 43.3% 16.75 95% Amnion 2.5% 1.361.5 Not 15.9 PBMC + calculated* 97.5% Amnion 1.25% 1.06 0.75 Not 12.27PBMC + calculated* 98.75% Amnion Notes: N/A—not applicable; *Notcalculated - values are close to the method detection limits

Example 18 Protein Array Analyses

The protein profiles of amniotic and chorionic membranes wereinvestigated using a SearchLight Multiplex chemiluminescent array. Thepresence of proteins in tissue membrane extracts and secreted by tissuesin culture medium was investigated. For comparison, two commerciallyavailable products containing living cells, Apligraf and Dermagraft,were assayed.

Example 18.1 Dermagraft

Dermagraft membrane was thawed and washed according to themanufacturer's instructions. Dermagraft membrane was cut into 7.5 cm²pieces. For tissue lysates, one 7.5 cm² piece of membrane was snapfrozen in liquid nitrogen followed by pulverization using a mortar andpestle. Crushed tissue was transferred to a 1.5 mL microcentrifuge tubeand 500 μL of Lysis buffer (Cell Signaling Technologies, Cat No. 9803)with protease inhibitor (Roche, Cat No. 11836153001) was added andincubated on ice for 30 min with frequent vortexing. The sample was thencentrifuged at 16000 g for 10 min. The supernatant was collected andsent for protein array analysis by Aushon Biosystems. For tissueculture, one 7.5 cm² piece of membrane was plated onto a well of a12-well dish and 2 mL of DMEM+1% HSA+ antibiotic/antimycotic were addedand incubated at 37° C.±2° C. for 3, 7, or 14 days. After incubation,tissue and culture media were transferred to a 15 mL conical tube andcentrifuged at 2000 rpm for 5 min. Culture supernatant was collected andsent for protein array analysis by Aushon Biosystems.

Example 18.2 Apligraf

Apligraf membrane was cut into 7.3 cm² pieces. For tissue lysates, one7.3 cm² piece of membrane was snap frozen in liquid nitrogen followed bypulverization using a mortar and pestle. Crushed tissue was transferredto a 1.5 mL microcentrifuge tube and 500 μL of Lysis buffer (CellSignaling Technologies, Cat No. 9803) with protease inhibitor (Roche,Cat No. 11836153001) was added and incubated on ice for 30 min withfrequent vortexing. The sample was then centrifuged at 16000 g for 10min. The supernatant was collected and sent for protein array analysisby Aushon Biosystems. For tissue culture, one 7.3 cm² piece of membranewas plated onto a well of a 12-well dish and 2 mL of DMEM+1% HSA+antibiotic/antimycotic were added and incubated at 37° C.±2° C. for 3,7, or 14 days. After incubation, tissue and culture media weretransferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5min. Culture supernatant was collected and sent for protein arrayanalysis by Aushon Biosystems.

Example 18.3 Amniotic and Chorionic Membranes

Amniotic and chorionic membranes were isolated and packaged at −80°C.±5° C. according to the manufacturing protocols disclosed herein inExample 3. Packaged membranes were then thawed in a 37° C.±2° C. waterbath and washed 3 times with DPBS. Membranes were cut into 8 cm² pieces.For tissue lysates, one 8 cm² piece of membrane was snap frozen inliquid nitrogen followed by pulverization using a mortar and pestle.Crushed tissue was transferred to a 1.5 mL microcentrifuge tube and 500μL of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) withprotease inhibitor (Roche, Cat No. 11836153001) was added and incubatedon ice for 30 min with frequent vortexing. Tissue lysate was thencentrifuged at 16000 g for 10 min. The supernatant was collected andsent for protein array analysis by Aushon Biosystems. For tissueculture, one 8 cm² piece of membrane was plated onto a well of a 12-welldish and 2 mL of DMEM+1% HSA+ antibiotic/antimycotic were added andincubated at 37° C.±2° C. for 3, 7, or 14 days. After incubation, tissueand culture media were transferred to a 15 mL conical tube andcentrifuged at 2000 rpm for 5 min. Culture supernatant was collected andsent for protein array analysis by Aushon Biosystems.

Initial testing consisted of an analysis of 36 proteins that areimportant for wound healing. The list of identified proteins isdescribed in Table 11.

TABLE 11 List of selected proteins for analysis. Protein Group Based onFunctionality Comments Metalloproteases Matrix Metalloproteinase 1Matrix and growth factor (MMP1), MMP2,3,7,8,9,10,13 degradation;facilitate cell migration. MMP Inhibitors Tissue Inhibitors of MMPs Haveangiogenic activity; (TIMP1 and 2) can be placed in the “angiogenicfactors” group. Angiogenic Factors Angiotensin-2 (Ang-2); basic Majorityof these factors Fibroblast Growth Factor also have growth and basic(bFGF); heparin-bound migration stimulatory Epidermal Growth Factoractivities and can be (HB-EGF); EGF; FGF-7 (also placed in a group ofknown as Keratinocyte growth factors. Growth Factor-KGF); Plateletderived Growth Factors (PDGF) AA, AB, and BB; Vascular EndothelialGrowth Factor (VEGF), VEGF-C and VEGF-D; Neutrophilgelatinase-associated lipocalin (NGAL); Hepatocyte Growth Factor (HGF);Placenta Growth Factor (PIGF); Pigment Epithelium Derived Factor (PEGF);Thrombopoetin (TPO) Protease Inhibitor/Protein Alpha-2-macroglobulinInhibit protease activity; Carrier regulate growth factor activity.Growth Factors See “angiogenic factors” + See “angiogenic factors.”Transforming Growth Factor alpha (TGF-a) Cytokines Adiponectin (Acrp-30)Affect keratinocyte functions. Granulocyte Colony- Protection frominfections. Stimulating Factor (G-CSF) Interleukin1 Receptor Regulateactivity of Antagonist (IL-1RA) inflammatory cytokine IL- 1. LeukemiaInhibitory Factor Support angiogenic (LIF) growth factors. ChemokinesSDF-1beta Attracts endothelial and other stem cells from circulation towound site. Regulators of IGF Insulin-like growth factor Regulate IGFactivity. binding protein (IGFBP1,2,3)

Example 18 Protein Expression in Present Placental Products

Preliminary protein array data analyses showed that the majority ofselected testing factors (refer to Table 11) were expressed in amnioticmembrane, chorionic membrane, Apligraf, and Dermagraft. Three proteinswere identified as unique for the amniotic membrane and/or the chorionicmembrane which are undetectable in Apligraf and Dermagraft. Theseproteins are EGF, IGFBP1, and Adiponectin. All three proteins areimportant for wound healing. FIG. 13 depicts expression of EGF (A),IGFBP1 (B), and Adiponectin (C) in amniotic or chorionic membranes. AM75and AM 78 are placental products of the present invention (e.g.cryopreserved), CM75 and CM78 are cryopreserved chorionic membraneproducts. These proteins are believed by the inventors to facilitate thetherapeutic efficacy of the present placental products for woundhealing.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising an amnioticmembrane containing EGF, IGFBP1, and/or adiponectin

Example 19 Wound Healing Proteins are Secreted for a Minimum of 14 Days

Placental products of the present invention demonstrate a durableeffect, desirable for wound healing treatments. The extracellular matrixand presence of viable cells within the amniotic membrane described inthis invention allow for a cocktail of proteins that are known to beimportant for wound healing to be present for at least 14 days. Amnioticmembranes were thawed and plated onto tissue culture wells and incubatedat 37° C.±2° C. for 3, 7, and 14 days. At each time point, a sample ofthe culture supernatant was collected and measured through protein arrayanalysis as described in Example 18. Table 12 illustrates the level ofvarious secreted factors in tissue culture supernatants from two donorsof amniotic membranes at 3, 7 and 14 days as measured through proteinarray analysis.

TABLE 12 Levels of proteins secreted in amnion tissue culturesupernatants at different time points (pg/ml). Day 3 Day 7 Day 14hACRP30 548.03 766.73 371.56 hAlpha2Macroglobulin 69687.55 31764.0048477.62 hANG2 0.00 9.28 1.65 hEGF 3.06 2.51 2.32 hbFGF 40.80 85.46269.97 hFibronectin 1932101.25 3506662.00 6019286.50 hHBEGF 41.78 80.5078.09 hHGF 5358.09 9327.67 18081.16 hIGFBP1 2654.57 6396.11 4666.88hIGFBP2 4379.76 23797.46 21784.21 hIGFBP3 36030.52 107041.71 13350.99hIL1ra 116593.20 675.09 4927.52 hKGF 7.29 13.86 36.59 hMMP1 323249.531727765.60 15272931.52 hMMP10 14804.44 20557.91 16194.56 hMMP13 92.92408.17 399.01 hMMP2 38420.90 322500.72 3283119.13 hMMP3 66413.54283513.74 3598175.53 hMMP7 128.51 147.65 4005.14 hMMP8 463.32 2109.212331.47 hMMP9 6139.53 25810.38 60483.67 hNGAL 15754.19 70419.63721923.09 hPDGFAA 18.02 58.69 16.31 hPDGFAB 16.58 58.41 28.30 hPDGFBB1.94 21.67 5.84 hPEDF 6793.74 21645.90 169990.84 hSDF1b 0.00 24.09 37.12hTGFa 15.05 14.89 205.90 hTGFb1 334.07 341.53 680.33 hTGFb2 119.59207.79 731.96 hTIMP1 197743.23 437492.21 247661.65 hTIMP2 4724.2519970.76 189810.51 hTSP1 0.00 0.00 1274.62 hTSP2 13820.61 59695.21991366.59 hVEGF 44.98 57.45 7.40 hVEGFC 548.03 766.73 371.56

Example 20 Interferon 2α (IFN-2α) and Transforming Growth Factor-β3(TGF-β3)

Placental products described in this invention have been analyzed forthe presence of IFN-2α and TGF-β3. Briefly, after thawing, the membraneswere homogenized and centrifuged at 16,000 g to collect the resultingsupernatants. Supernatants were analyzed on a commercially availableELISA kit from MabTech (IFN-2α) and R&D Systems (TGF-β3). FIG. 14 showssignificant expression of IFN-2α (A) and TGF-β3 (B) in placental producthomogenates.

Without being bound by theory, interferon-2α and TGF-β3 may aid in theprevention of scar and contracture formation. IFN-2α may serve a role todecrease collagen and fibronectin synthesis and fibroblast-mediatedwound contracture.

Example 21 Tissue Reparative Proteins in Amniotic Membranes

Placental product homogenates were analyzed for the presence of proteinsthat are important in tissue repair.

Placental product described in this invention have been analyzed for thepresence of tissue reparative proteins. Briefly, the thawed productswere incubated in DMEM+10% FBS for 72 hrs. The membranes were thenhomogenized in a bead homogenizer with the culture media. Thehomogenates were centrifuged, and the supernatants were analyzed oncommercially available ELISA kits from R&D Systems. FIG. 15 showssignificant expression of BMP-2, BMP-4, PLAB, PIGF, and IGF-1 in severaldonors of amniotic membranes.

Without being bound by theory, the inventors believe that efficacy ofthe present placental products for wound repair are due, in part, to therole of BMPs, IGF-1, and PIGF in the development and homeostasis ofvarious tissues by regulating key cellular processes. BMP-2 and BMP-4may stimulate differentiation of MSCs to osteoblasts in addition topromote cell growth; placental BMP or PLAB is a novel member of the BMPfamily that is suggested to mediate embryonic development. Insulin-likegrowth factor 1 (IGF-1) may promotes proliferation and differentiationof osteoprogenitor cells. Placental derived growth factor (PIGF) mayacts as a mitogen for osteoblasts.

Example 22 MMPs and TIMPs

Both MMPs and TIMPs are among the factors that are important for woundhealing. However, expression of these proteins must be highly regulatedand coordinated. Excess of MMPs versus TIMPS is a marker of poor chronicwound healing. We investigated expression of MMPs and TIMPs and itsratio in amniotic membrane and chorionic membrane and compared it to theexpression profile in Apligraf and Dermagraft.

Results showed that all membranes express MMPs and TIMPs; however, theratio in the thawed placental products and chorionic membranes issignificantly lower. Therefore, these membranes will be more beneficialfor wound healing (FIG. 16).

Accumulated data indicate that the MMP to TIMP ratio is higher in casesof non-healing wounds. For example, the ratio between MMP-9 and TIMP1 isapproximately 7-10 to one for good healing and 18-20 or higher for poorhealing. Analysis of the ratio between MMPs and TIMPs secreted byplacental tissues, Apligraf, and Dermagraft showed that the amniotic andchorionic membrane products contain MMPs and TIMPs at an approximateratio of 7, which is favorable for wound healing. In contrast,Dermagraft had a ratio>20, and Apligraf had a ratio>200.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising an amnioticmembrane containing MMP-9 and TIMP1 at a ratio of about 7-10 to one.

Example 23 α2-Macroglobulin

α2-macroglobulin is known as a plasma protein that inactivatesproteinases from all 4 mechanistic classes, serine proteinases, cysteineproteinases, aspartic proteinases, and metalloproteinases. Anotherimportant function of this protein is to serve as a reservoir forcytokines and growth factors, examples of which include TGF, PDGF, andFGF. In the chronic wounds like diabetic ulcers or venous ulcers, thepresence of high amount of proteases leads to rapid degradation ofgrowth factors and delays in wound healing. Thus, the presence ofα2-macroglobulin in products designed for chronic wound healing will bebeneficial. Results of the protein array analysis showed that amnioticand chorionic membranes contain α2-macroglobulin (Table 13). Althoughthese preliminary data show high variability between donors, theimportance of this protein in wound healing prompted the additionalevaluation of α2-macroglobulin in placental tissues using a singleanalyte ELISA instead of protein array, which is a useful tool toevaluate the presence of multiple proteins in one sample for profiling.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising an amnioticmembrane containing α2-macroglobulin.

TABLE 13 Expression of α2-macroglobulin in placental tissue proteinextracts. α2-macroglobulin Sample (pg/mL/cm²) AM75 7 CM75 790 AM78 53042CM78 1014

Example 24 Establishment of EGF as a Marker for Amniotic Tissue Potency

EGF is among the factors that are important for wound healing (Schultzet al., 1991, Komarcevic, 2000, and Hong et al., 2006). The absence ordecreased amount of EGF is one characteristic of chronic wounds (Hardinget al., 2002). Evaluation of proteins derived from amniotic membranesamples prepared according to the developed manufacturing processdisclosed by the present application reveal that EGF is one of the majorfactors secreted in higher quantities by these tissues. The importanceof EGF for wound healing together with high levels of EGF detected inthe presently disclosed amniotic membranes support selection of EGF as apotency marker for evaluation of membrane products manufactured forclinical use pursuant to the present disclosure. A commerciallyavailable ELISA kit from R&D Systems was selected for evaluation of itssuitability to measure EGF secreted by amniotic membranes. ELISAqualification meets the standards established by the FDA and ICHguidances for bioanalytical assay validation (Validation of AnalyticalProcedures: Text and Methodology Q2(R1), 1994; ICH Harmonized TripartiteGuideline and Guidance for Industry Bioanalytical Method Validation,2001). Amniotic membranes evaluated for expression of EGF by this methodconfirmed protein array data and further demonstrated that EGF was aunique factor expressed at clinically significant levels in thesetissues.

Example 25 Amniotic Tissue Expression of EGF

Protein array analysis provided initial evidence that EGF was uniquelyexpressed in amniotic membranes but not in chorionic membranes (Table14). The levels of EGF measured in amniotic membranes were of clinicalsignificance.

TABLE 14 Protein array data showing range of expression of EGF inamniotic and chorionic membranes from multiple donors. Amnion Chorion(pg/ml) (pg/ml) EGF 127.3-361.4 0-0.8

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising an amnioticmembrane containing EGF, optionally in substantial amounts.

Homogenate of Placental Products

Placental products were thawed until no remaining frozen cryomedia waspresent. Membranes were then removed from bags and cut into 4 cm×2 cmpieces while still adhered to nitrocellulose. Each piece of tissue wasthen removed from the nitrocellulose and washed twice with PBS. Eachtissue was then snap frozen in a homogenization tube using liquidnitrogen. Subsequently, one pre-cooled 5 mm steel bead was added to eachtube; samples were then homogenized using a Qiagen Tissue Lyseraccording to the manufacturer's recommendations in 500 μL homogenizationmedia. Tissue homogenates were stored at −80° C.±5° C. until analyzed byELISA for EGF expression.

ELISA Procedure and Validation

Samples were analyzed for the expression of EGF using the QuantikineHuman EGF ELISA Kit (R&D Systems) according to the manufacturer'srecommendations. Several parameters were tested to establish the testcriteria and to show the suitability of this ELISA kit to measure EGF inplacental tissue samples. Assay performance was assessed by analyzinglinearity, range, lower and upper limits of quantitation (LLOQ andULOQ), precision, accuracy, and robustness. Experimental data (Table 14)showed that the quantitation range of this assay was 3.9-250 pg/mL EGF.The intra- and inter-assay CVs ranged from 1.22 to 5.80% and 2.73 to7.53%, respectively. Additionally, sample recovery analysis demonstratedaccuracy within 20%. Furthermore, this assay showed dilutional linearityand specificity. Ruggedness was also demonstrated by assay insensitivityto variations introduced by different analysts.

TABLE 15 Established ELISA parameters for measuring EGF in placentahomogenates. Calibration Standard 3.9-250 pg/mL Range Assay Quantitation7.8-250 pg/mL Range LLOQ 7.8 pg/mL LOD 2.18 pg/mL ULOQ 250 pg/mLEGF Expression in Amniotic Membranes

Measurement of EGF in amniotic preparations has proven to be bothreliable and reproducible. Measurement of EGF in multiple donors showedthat this method of quantification was a valuable means of evaluatingpotency in tissue prepared pursuant to the present disclosure for use ina clinical setting. FIG. 17 shows representative expression of EGF in athawed placental product and chorionic membrane prepared and analyzed bythe methods described above. Results have been reproduced in multipletissue preparations.

These data are consistent with certain embodiments of the presentinvention that provide a placental product comprising an amnioticmembrane containing EGF.

Example 26 Placental Tissues Enhance Cell Migration and Wound Healing

The process of wound healing is highly complex and involves a series ofstructured events controlled by growth factors (Goldman, 2004). Theseevents include increased vascularization, infiltration by inflammatoryimmune cells, and increases in cell proliferation. The beginning stagesof wound healing revolve around the ability of individual cells topolarize towards the wound and migrate into the wounded area in order toclose the wound area and rebuild the surrounding tissue. Upon properstimulation, several different types of cells including epithelial,endothelial, mesenchymal, and fibroblastic cells are implicated in thewound healing process (Pastar et al, 2008 and Bannasch et al., 2000).Specifically, they proliferate and migrate into the wound area topromote healing. Therefore, experiments were conducted to determine iffactors secreted from amniotic and chorionic membranes produced pursuantto the present disclosure promote cell migration and wound fieldclosure. To accomplish this, a commercially available wound healingassay (Cell Biolabs) and a highly accepted human microvascularendothelial cell line (HMVEC, Lonza Inc.) were utilized. Resultsindicated that cell migration was enhanced by treatment with conditionedmedia from the placental membranes.

In Vitro Cell Migration Assay

Human microvascular endothelial cells (HMVECs) were grown under normalcell culture conditions in defined complete media (Lonza Inc.). Toassess migration and wound field closure, a commercially available woundhealing assay was used (Cell Biolab).

FIG. 18 depicts the Cell Biolabs 24-well Cytoselect wound healing assay.(Figure reproduced from Cell Biolabs).

Cells were collected via trypsinization, pelleted, and counted beforebeing resuspended in complete media at a density of 2×10⁵ cells/mL. 250μL (5×10⁴ cells) of cell suspension was then pipetted into each side ofa well containing a wound healing insert (Cytoselect 24-well WoundHealing Assay Plate, Cell Biolabs). The cells were grown for 24 hours incomplete media. After 24 hours, the wound inserts were removed. At thesame time, complete media was removed and replaced with experimentalmedia. Complete media and basal media were used as positive and negativecontrols, respectively. To generate experimental media, placentalmembranes were incubated for 3 days in DMEM with 1% human serum albumin(HSA) in a tissue culture incubator. The resulting tissue and media werethen placed in eppendorf tubes and spun at high speed in amicrocentrifuge. The supernatants were collected and stored at −80°C.±2° C. until use. For migration and wound healing studies, conditionedmedia from placental membranes was diluted 1:20 in basal media beforebeing added to experimental wells. After 18 hours, the media wasremoved, and the cells were fixed for 20 min in 4% paraformaldehyde andstained with crystal violet. The wound field in each well was thenphotographed. Wound healing was determined by the amount of wound fieldstill visible at the end of the experiment when compared to controlpictures taken before conditioned media was added to the wells.

Placental Membrane Conditioned Media Supports Cell Migration and WoundField Closure

Conditioned media from amniotic and chorionic membranes was used toassess the potential for these membranes to promote cell migration andwound field closure. Conditioned media from placental amniotic,chorionic, and a combination of amniotic/chorionic membranes supportedmigration of cells into the experimental wound field.

FIG. 19 depicts representative images of cells treated with 5%conditioned media from amniotic, chorionic, or a combination ofamniotic/chorionic tissue as well as positive and negative controls.Wound field is 0.9 mm in width.

The ability of factors from placental membranes produced pursuant to thepresent disclosure to promote HMVEC migration indicated that thesetissues have the ability to enhance wound healing. Additionally, basedon the insight of the inventors, it has been surprisingly discoveredthat these tissues also enhance revascularization since the HMVEC cellline is derived from vascular endothelial cells.

These data demonstrate that placental products of the present inventionproduce unexpectedly superior levels of factors that promote woundhealing therapies.

Example 27 Biochemical Profile of the Supernatants from ExemplaryPlacental Tissue Products

Table 16 depicts the biochemical profile of the supernatants ofexemplary placental products of the invention (results adjusted per cm²after subtraction of the negative background).

TABLE 16 Factors in Placental Tissue Product (pg/cm²). Units ApligrafDermagraft AM75 CM75 AM78 CM78 hMMP1 pg/ml/cm² 1964945.37 14818.202821.85 3531.81 117326.89 95.46 hMMP7 pg/ml/cm² 911.54 0.00 0.00 0.003.96 0.00 hMMP10 pg/ml/cm² 0.00 0.00 113.94 0.00 0.00 0.00 hMMP13pg/ml/cm² 21.61 0.00 0.00 0.00 0.71 0.00 hMMP3 pg/ml/cm² 208281.70180721.52 170.26 161.52 8325.17 0.00 hMMP9 pg/ml/cm² 8872.28 19321.39214.78 1455.11 630.56 57.59 hMMP2 pg/ml/cm² 153341.77 19712.21 287.1437.93 3823.38 24.44 hMMP8 pg/ml/cm² 36.92 12.19 0.00 0.00 0.00 0.00hTIMP1 pg/ml/cm² 2487.18 10909.84 569.23 883.05 28743.48 97.94 hTIMP2pg/ml/cm² 7285.53 1796.56 89.29 13.72 424.06 4.83 MMP/TIMP 239.26 19.726.81 6.26 4.50 2.62

Example 28 Biochemical Profile of the Lysates from Exemplary PlacentalTissue Products

Table 17 depicts the biochemical profile of the lysates of exemplaryplacental tissue products of the invention (results adjusted per cm²after subtraction of the negative background).

TABLE 17 AM75 lysate AM78 lysate CM75 lysate CM78 lysate pg/ml pg/mlpg/ml pg/ml hACRP30 50.8 1154.6 1213.7 225.3 hAlpha2- 1910.6 426191.68174.4 9968.6 Macroglobulin hEGF 127.3 361.4 0.0 0.8 hbFGF 119.1 821.5375.0 351.3 hGCSF 0.7 3.2 1.2 0.7 hHBEGF 127.5 168.0 15.4 84.5 hHGF3943.7 15060.0 29979.6 50392.8 hIGFBP1 5065.0 9456.6 934.0 1443.6hIGFBP2 12460.8 5569.7 135.9 134.6 hIGFBP3 50115.7 41551.4 4571.511970.2 hIL1ra 3881.0 32296.9 5168.2 525.5 hKGF 1.4 8.8 3.1 1.5 hLIF 0.04.2 0.0 0.0 hMMP1 9144.1 20641.2 2882.9 6582.3 hMMP10 0.0 15.5 79.3 87.5hMMP2 2067.3 4061.9 949.5 748.8 hMMP3 0.0 36.2 0.0 0.0 hMMP7 5.1 11.44.5 9.1 hMMP8 0.0 0.0 0.0 0.0 hMMP9 92.2 2878.1 2676.2 1259.3 hNGAL6900.1 6175.9 938.5 229.7 hPDGFAA 0.0 12.5 39.8 35.2 hPDGFAB 11.2 31.314.4 14.0 hPDGFbb 4.6 13.4 4.0 1.3 hPEDF 0.0 652.6 0.0 0.0 hTIMP1 7958.135955.6 50712.3 17419.9 hTIMP2 3821.8 7443.2 640.7 780.0 hVEGF 3.3 11.8125.2 8.4 hVEGFC 46.5 150.0 123.5 51.7 hVEGFD 25.7 31.0 15.0 20.4

Example 29 Use of Placental Products for Treating Diabetic Foot Ulcers

Purpose: Despite bioengineered skin substitutes that contain humanfibroblasts or a combination of human fibroblasts and keratinocytes,published rates of chronic wound healing remain low, with approximatelyhalf of all wounds recalcitrant to even these newer therapies. Morbidityand mortality from diabetic foot ulceration are substantial as the 5year mortality rate following a lower extremity amputation is between39% and 68%. (Page J. J of Foot & Ankle Surgery 2002; 41(4):251-259;Isumi Y., et al. Diabetes Res and Clin Practice 2009; 83:126-131).

A instant membrane product, which provides necessary angiogenic andanti-inflammatory growth factors was introduced in an effort to improveoutcomes of patients with chronic skin ulceration at-risk foramputation.

Objective: Patients with chronic diabetic foot ulceration, unresponsiveto available therapy and at-risk for amputation were considered fortreatment. All wounds were aggressively debrided prior to graftapplication. Patients were evaluated regularly and application of amembrane product of the present invention was at the discretion of thetreating physician. Offloading was encouraged in both patients.

Introduction

According to the United States Food and Drug Administration (FDA), achronic, cutaneous ulcer is defined as a wound that has failed toproceed through an orderly and timely series of events to produce adurable structural, functional and cosmetic closure (2). The most commonchronic wounds include pressure ulcers and leg ulcers. The triad ofperipheral neuropathy, deformity, and minor trauma has emerged as themost frequent causes of insult that lead to foot ulcerations. In termsof healing rates, an appropriate benchmark for a chronic wound is adecrease of 10% to 15% in size every week, or 50% decrease in size overa one-month period. A three-year retrospective cohort study performed byRamsey et al. of 8,905 patients in a large health maintenanceorganization who have diabetes reported a 5.8% cumulative incidence ofulceration. At the time of diagnosis, 15% of these patients developedosteomyelitis and 16% required partial amputation of a lower limb.

Approximately 80% to 85% of lower extremity amputations are preceded byfoot ulcerations. Morbidity and mortality from diabetic foot ulcerationare substantial as the 5 year mortality rate following a lower extremityamputation is between 39% and 68%. (2). These mortality rates are higherthan the five-year mortality rates for breast cancer, colon cancer, andprostate cancer.

Despite all of the advances in bioengineered tissue for the treatment ofchronic diabetic ulcerations, there are an abundance of patients whoseulcerations are resistant to therapy, and result in a chronic wound.Because of healing rates that only approach 50% with these newertherapies, the use of stem cells in regenerative medicine has been ofparticular interest recently. The ultimate aim is to promote restorationof functional skin. A preliminary study was performed by Fiami et al. inwhich they isolated mesenchymal stem cells from umbilical cord blood andinoculated them onto a piece of de-epithelialized dermis. The results ofthis preliminary study showed that peripheral stem cells are capable ofsurviving and expressing neoangiogenesis. In addition to showing promisefor tissue repair, mesenchymal stem cells exhibit low immunogenicity andcan be transplanted universally without having to undergo compatibilitytesting between the donor and recipient.

In this study, clinical evidence of remarkable healing using an instantmembrane product for the treatment of two chronic wounds that amputationwas considered. The fundamentals of wound management are still thecornerstone of comprehensive wound care in any treatment protocolincluding adequate debridement, offloading, maintaining a moistenvironment, and adequate perfusion and infection control.

Materials

An instant membrane product was made as taught herein, comprising anallograft derived from the aminon comprising a bilayer of nativeepithelial cells on a basement membrane and a stoma layer consisting ofneonatal fibroblasts, extracellular matrix (ECM) and mesenchymal stemcells (MSC).

Limb Salvage: Case One

History and Physical Examination

A 70 year old male presented to the emergency department with bullaformation on the dorsolateral aspect of his right foot between thefourth and fifth digits, edema and pain, and a small lesion lateral tothe fifth digit. The patient reported a history of minor trauma to thearea two weeks prior to presentation. The patient had a history of typeII diabetes mellitus, hypertension, heart failure, chronic obstructivepulmonary disease, and chronic kidney disease treated with hemodialysisthree times a week. The patient had a surgical history of anaorta-venous graft replacement. He denied any history of alcohol,tobacco or drug use. Physical exam revealed no active purulent drainageor malodor, and no tenderness on palpation. The vascular exam revealednon-palpable pulses in the dorsalis pedis and posterior tibial arteries.Doppler exam revealed a monophasic dorsalis pedis pulse with a biphasicposterior tibial artery pulse. The fifth digit had gangrenous changesand was cold on palpation. There were ischemic changes of the fourthdigit. Radiographic evaluation revealed scattered air densitiesindicative of soft tissue gas in the fourth interspace as well as thetip of the fifth digit.

Preoperative Management

The patient was started on intravenous antibiotics of vancomycin andpiperacillin and tazobactam at appropriate renal dosing.

Operative Management

The patient was taken to the operating room where an incision anddrainage of the fourth interspace was performed, and a partial fifth rayamputation to the level of the metatarsal head was performed withoutcomplication. The wound was left open and packed with sterile gauzemoistened with sterile normal saline, and covered with a sterilecompressive dressing. Intraoperative findings revealed liquefactivenecrosis of surrounding tissues with purulence and malodor. The patientunderwent 2 subsequent surgical debridements, with the second resultingin further removal of the fourth and fifth metatarsal shafts. In a thirdsurgery further debridement of necrotic soft tissue and amputation ofthe fourth digit was performed. On May 20, 2010 treatment with aninstant membrane product was initiated. Prior to the graft placement thepatient had undergone successful recanalization of the popliteal arteryand the peroneal artery without significant residual stenosis.

Postoperative Course

The patient followed up with his podiatric surgeon within 2 days ofbeing discharged from the hospital. Upon initial exam, there were noclinical signs of infection, and the proximal dorsal incision appearedcoapted. The third digit was dusky and cool in appearance. Radiographswere taken which showed no evidence of soft tissue gas or acuteosteomyelitis. A dry sterile dressing was applied. The patient receivedapplications of the insant membrane product at 6 additional visits in anoutpatient office. Prior to each application the wound was evaluated forabscess, cellulitis, drainage, hematoma formation, and infection. Ateach visit, the wound decreased in size and appeared more granular innature as compared to previous visits. At the time of the thirdapplication the wound had decreased in size 50%.

At 19 weeks the wound was considered closed, and the patient wasinstructed to remain weight bearing on the affected limb with the use ofa surgical shoe only.

Photographs of the remarkable wound healing mediated by a placentalproduct of the present invention as shown in FIG. 20. Panel A: Firstapplication of an instant membrane product; B: 8 weeks post first aninstant membrane product application; C: 10½ weeks post first an instantmembrane product application; D: 12 weeks post first an instant membraneproduct application; E: 19 weeks post first an instant membrane productapplication.

Limb Salvage: Case Two

History and Physical Examination

A 44-year old male presented to an outpatient office with a largeulceration on the plantar aspect of his left hallux, secondary to aprevious trauma a few weeks prior to the visit. The patient had ahistory of diabetes mellitus for the past five years complicated byperipheral neuropathy, hypertension, dyslipidemia, and osteomyelitis.Past surgical history included abdominal aortic aneurysm repair andcircumcision. On physical exam the ulceration measured 4.0 cm×2.0 cm×1.5cm, probing to the distal phalanx with exposed tendon. There was noascending cellulitis or lymphangitis, and no increased temperaturegradient. Capillary fill time, hair growth, and tissue turgor were allnormal. There were palpable pulses in the dorsalis pedis and theposterior tibial artery. Radiographic exam was negative for soft tissuegas. Magnetic resonance imaging revealed osteomyelitis in the distalaspect of the proximal phalanx and the distal phalanx of the great toewith a small soft tissue abscess in the region of the dorsal soft tissueadjacent to the distal phalanx.

Preoperative Management

The patient was started on intravenous antibiotics. He was taken to theoperating room for excisional debridement of all nonviable tissue andapplication of the instant membrane product.

Operative Management

The ulceration was debrided to healthy tissue with utilization of bothsharp dissection and Versajet™, leaving the head of the proximal phalanxexposed plantarly. The instant membrane product was then placed over thewound bed and exposed bone. The patient tolerated the procedure withoutcomplication. The patient was discharged from the hospital the day aftersurgery on a five week course of intravenous antibiotic therapy.

Postoperative Course

The patient was instructed to remain strictly non-weight bearing to theaffected limb, and returned for follow-up on post-operative day 6. Thedressing was clean, dry and intact. There were no post-operativecomplications such as abscess, cellulitis, discomfort, or drainage andno clinical signs of infection. The patient received a total of 7 stemcell graft applications over the course of the next 8 weeks. At eachvisit the wound was inspected for clinical signs of infection.Evaluation at each visit revealed marked development in granulationtissue to the wound base and significant decrease in size. Eight weeksafter the initial application of the allograft tissue the wound wasclosed.

Photographs of the remarkable wound healing mediated by a placentalproduct of the present invention as shown in FIG. 21. Panel A:Osteomyelitis, tendon exposed, probed to bone. First stem cell graft wasapplied after surgical debridement; B: Status post 1 application of stemcell graft, wound is granular in nature and no signs of infection; C: 3weeks post surgical intervention; 2 applications on the instant membraneproduct, the wound is considerably smaller in circumference and depth;D: 6 weeks post surgical intervention the wound is almost closed; E: 8weeks and 7 applications of the instant membrane product, the wound isclosed.

CONCLUSION

Despite the tremendous progress in skin tissue engineering in the pastfew decades, current therapy has limited efficacy in the treatment ofchronic diabetic ulceration. As shown in this case report of twopatients, the use of advanced therapies containing stem cells may proveuseful to ultimately heal these patients in lieu of amputation, reducemortality rates, and at the same time be a cost effective alternative tostandard treatments currently on the market. Both patients highlightedin this case report received 7 applications of a membrane product of thepresent invention. Complete healing occurred in both patients. Therewere no reported complications associated with treatment; the instantmembrane product was safe and effective in an initial evaluation of twopatients with diabetic foot ulceration at-risk for amputation. Theseresults indicate that patients with recalcitrant, chronic wounds shouldbe considered for this novel therapy.

The invention claimed is:
 1. A cryopreserved amniotic membranecomprising viable cells native to the amniotic membrane; wherein thecryopreserved amniotic membrane is immunocompatible; wherein at least70% of the cells are viable; wherein the viable cells comprise two ormore cell types consisting of MSCs, fibroblasts, and epithelial cells;wherein the cryopreserved amniotic membrane comprises less than about 1%of CD14+ macrophages.
 2. The cryopreserved amniotic membrane of claim 1,wherein the cryopreserved amniotic membrane comprises one or moretherapeutic factors native to the amniotic membrane.
 3. Thecryopreserved amniotic membrane of claim 1, wherein the cryopreservedamniotic membrane comprises extracellular matrix that is native to theamniotic membrane.
 4. The cryopreserved amniotic membrane of claim 1,wherein the membrane is immunocompatible by being selectively depletedin one or more types of functional immunogenic cells.
 5. Thecryopreserved amniotic membrane of claim 1, wherein the amnioticmembrane has a substantial decrease in TNF-α release afterlipopolysaccharide (LPS) stimulation.
 6. The cryopreserved amnioticmembrane of claim 5, wherein the amniotic membrane releases TN F-α in anamount less than 420 pg/ml.
 7. The cryopreserved amniotic membrane ofclaim 1, wherein the amniotic membrane is immunocompatible by beingsubstantially free of immunogenic maternal cells.
 8. The cryopreservedamniotic membrane of claim 7, wherein the immunogenic maternal cells areselected from the group consisting of maternal dendritic cells, maternalleukocytes, and a combination thereof.
 9. The cryopreserved amnioticmembrane of claim 1, wherein the viable cells further comprise stromalcells.
 10. The cryopreserved amniotic membrane of claim 9, wherein thestromal cells are in an amount of about 2,000 to about 15,000 per cm² ofthe amniotic membrane.
 11. The cryopreserved amniotic membrane of claim1, wherein the cryopreserved amniotic membrane is associated with anitrocellulose substrate/support.
 12. The cryopreserved amnioticmembrane of claim 1, further comprising at least one cell-permeatingcryopreservative, at least one non-cell permeating cryopreservative,derivatives thereof or a combination thereof.
 13. The cryopreservedamniotic membrane of claim 12, wherein the cell permeatingcryopreservative, the non-cell-permeating cryopreservative, derivativethereof, or combination thereof is present in an amount of about 5% toabout 20% by volume.
 14. The cryopreserved amniotic membrane of claim13, wherein the cryopreservative comprises DMSO.
 15. The cryopreservedamniotic membrane of claim 1, wherein the amniotic membrane comprisesMSCs and at least 40% of the MSCs are viable after a freeze-thaw cycle.16. A previously cryopreserved membrane comprising an amniotic membrane,wherein the previously cryopreserved amniotic membrane comprises: a)viable cells, wherein said viable cells are native to the amnioticmembrane before cryopreservation and greater than 70% of said cells areviable, wherein the viable cells comprise two or more cell typesconsisting of MSCs, fibroblasts, and epithelial cells; b) one or moretherapeutic factors that are native to the amniotic membrane; c)extracellular matrix that is native to the amniotic membrane; and d)less than about 1% of CD14+ macrophages.
 17. The cryopreserved amnioticmembrane of claim 1, wherein the amniotic membrane comprises anepithelial layer, a basement membrane layer, and a stromal layer,wherein all layers are from the same amniotic membrane.
 18. Thecryopreserved amniotic membrane of claim 17, wherein the amnioticmembrane comprises fibroblasts in an amount of about 2% to about 30% oftotal cells, and wherein at least 40% of the fibroblasts are viable. 19.The cryopreserved amniotic membrane of claim 17, wherein the amnioticmembrane comprises MSCs in an amount of at least about 1% to about 10%of the total cells.
 20. The cryopreserved amniotic membrane of claim 17,wherein the amniotic membrane comprises epithelial cells in an amount ofat least about 40% to about 90% of the total cells.